Stabilized formulations containing anti-PCSK9 antibodies

ABSTRACT

The present invention provides pharmaceutical formulations comprising a human antibody that specifically binds to human proprotein convertase subtilisin/kexin type 9 (PCSK9). The formulations may contain, in addition to an anti-PCSK9 antibody, at least one amino acid, at least one sugar, or at least one non-ionic surfactant. The pharmaceutical formulations of the present invention exhibit a substantial degree of antibody stability after storage for several months.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/559,862, filed on Jul. 27, 2012, which claims the benefit under 35USC §119(e) of U.S. Provisional Patent Application No. 61/512,666, filedon Jul. 28, 2011, the contents of which are herein specificallyincorporated by reference in their entireties.

FIELD

The present invention relates to the field of therapeutic antibodyformulations. More specifically, the present invention relates to thefield of pharmaceutical formulations comprising a human antibody thatspecifically binds to human proprotein convertase subtilisin/kexin type9 (PCSK9).

SEQUENCE LISTING

An ST.25 compliant text file of a sequence listing is filed concurrentlywith the present specification. The contents of the text file are hereinincorporated by reference. A paper copy of the sequence listing, whichis identical in content to the ST.25 compliant text file, is included aspart of the present specification.

BACKGROUND

Therapeutic macromolecules (e.g., antibodies) must be formulated in amanner that not only makes the molecules suitable for administration topatients, but also maintains their stability during storage andsubsequent use. For example, therapeutic antibodies in liquid solutionare prone to degradation, aggregation or undesired chemicalmodifications unless the solution is formulated properly. The stabilityof an antibody in liquid formulation depends not only on the kinds ofexcipients used in the formulation, but also on the amounts andproportions of the excipients relative to one another. Furthermore,other considerations aside from stability must be taken into accountwhen preparing a liquid antibody formulation. Examples of suchadditional considerations include the viscosity of the solution and theconcentration of antibody that can be accommodated by a givenformulation, and the visual quality or appeal of the formulation. Thus,when formulating a therapeutic antibody, great care must be taken toarrive at a formulation that remains stable, contains an adequateconcentration of antibody, and possesses a suitable viscosity as well asother properties which enable the formulation to be convenientlyadministered to patients.

Antibodies to the human proprotein convertase subtilisin/kexin type 9protein (PCSK9) are one example of a therapeutically relevantmacromolecule that requires proper formulation. Anti-PCSK9 antibodiesare clinically useful for the treatment or prevention of diseases suchas hypercholesterolemia and other dyslipidemias, and other conditions.Exemplary anti-PCSK9 antibodies are described, inter alia, in WO2008/057457, WO 2008/057458, WO 2008/057459, WO 2008/063382, WO2008/125623, U.S. Pat. No. 7,572,618, WO 2010/077854, US 2010/0166768,and US 2011/0065902.

Although anti-PCSK9 antibodies are known, there remains a need in theart for novel pharmaceutical formulations comprising anti-PCSK9antibodies that are sufficiently stable and suitable for administrationto patients.

SUMMARY

The present invention satisfies the aforementioned need by providingpharmaceutical formulations comprising a human antibody thatspecifically binds to human proprotein convertase subtilisin/kexin type9 protein (PCSK9).

In one aspect, a liquid pharmaceutical formulation is provided,comprising: (i) a human antibody that specifically binds to humanproprotein convertase subtilisin/kexin type 9 protein (PCSK9); (ii) abuffer; (iii) an organic cosolvent; (iv) a stabilizer; and optionally(v) a viscosity reducer.

In one embodiment, the antibody is provided at a concentration fromabout 50±7.5 mg/mL to about 200±30 mg/mL. In another embodiment, theantibody is provided at a concentration of about 50 mg/ml±7.5 mg/mL. Inanother embodiment, the antibody is provided at a concentration of about100 mg/mL±15 mg/mL. In another embodiment, the antibody is provided at aconcentration of about 150 mg/mL±22.5 mg/mL. In another embodiment, theantibody is provided at a concentration of about 175 mg/mL±26.25 mg/mL.In another embodiment, the antibody is provided at a concentration ofabout 200 mg/mL±30 mg/mL.

In one embodiment, the antibody comprises any one or more of an aminoacid sequence of SEQ ID NO:1-8. In one embodiment, the antibodycomprises (a) a heavy chain variable region (HCVR) comprising heavychain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3)each comprising a sequence of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4,respectively; and (b) a light chain variable region (LCVR) comprisinglight chain complementarity determining regions 1, 2 and 3(LCDR1-LCDR2-LCDR3) each comprising a sequence of SEQ ID NO:6, SEQ IDNO:7 and SEQ ID NO:8, respectively. In a specific embodiment, theantibody comprises an HCVR and an LCVR, each of which comprises theamino acid sequence of SEQ ID NO:1 and SEQ ID NO:5, respectively.

In one embodiment, the pH of the liquid formulation is about pH 6.0±0.5,pH 6.0±0.4, pH 6.0±0.3, pH 6.0±0.2, pH 6.0±0.1, pH 6.0±0.05, pH6.0±0.01, or pH 6.0. In a specific embodiment, the pH of the liquidformulation is about pH 6.0±0.3. In one embodiment, the liquidpharmaceutical buffer comprises one or more buffers, which has aneffective buffering range of about pH 5.5 to about pH 7.4, or a pKa ofabout 6.0.

In one embodiment, the buffer is histidine. In one embodiment, thehistidine is at a concentration of 5 mM±0.75 mM to 50 mM±7.5 mM. In oneembodiment, the histidine is at a concentration of 10 mM±1.5 mM or about10 mM. In one embodiment, the histidine is at a concentration of 20 mM±3mM or about 20 mM. In one embodiment, the histidine is at aconcentration of 40 nM±6 mM or about 40 nM.

In one embodiment, the organic cosolvent is a nonionic polymercontaining a polyoxyethylene moiety. In some embodiments, the organiccosolvent is any one or more of polysorbate 20, poloxamer 188 andpolyethylene glycol 3350. In a specific embodiment, the organiccosolvent is polysorbate 20.

In one embodiment, the organic cosolvent is at a concentration of fromabout 0.005%±0.00075% to about 1%±0.15% “weight to volume” or “w/v”,wherein, e.g., 0.1 g/ml=10% and 0.01 g/ml=1%. In one embodiment, theorganic cosolvent is polysorbate 20, which is at a concentration ofabout 0.2%±0.03% w/v. In another embodiment, the organic cosolvent ispolysorbate 20, which is at a concentration of 0.01%±0.0015% w/v orabout 0.01% w/v.

In one embodiment, the stabilizer is a sugar. In one embodiment, thesugar is selected from the group consisting of sucrose, mannitol andtrehalose. In a specific embodiment, the stabilizer is sucrose.

In one embodiment, the stabilizer is at a concentration of from 1%±0.15%w/v to 20%±3% w/v. In a specific embodiment, the stabilizer is sucroseat a concentration of 5%±0.75% w/v or about 5% w/v. In another specificembodiment, the stabilizer is sucrose at a concentration of 10%±1.5% w/vor about 10% w/v. In another specific embodiment, the stabilizer issucrose at a concentration of 12%±1.8% w/v or about 12% w/v.

In one embodiment, the viscosity reducer is a salt selected from thegroup consisting of arginine hydrochloride, sodium thiocyanate, ammoniumthiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zincchloride and sodium acetate. In one embodiment, the viscosity reducer isL-arginine hydrochloride.

In one embodiment, the viscosity reducer is at a concentration of from10 mM±1.5 mM to 150 mM±22.5 mM. In one embodiment, the viscosity reduceris L-arginine hydrochloride at a concentration of 50 mM±7.5 mM or about50 mM. In one embodiment, the viscosity reducer is L-argininehydrochloride at a concentration of 40 mM±6 mM or about 40 mM.

In one embodiment, the viscosity of the liquid or reconstitutedlyophilized pharmaceutical formulation at 25° C. is less than or equalto about 15 cPoise±10%. In one embodiment, the viscosity at 25° C. isbetween 1.0 cPoise±10% and 18 cPoise±10%. In one embodiment, theviscosity at 25° C. is 1.6 cPoise±10%, 1.7 cPoise±10%, 3.3 cPoise±10%,3.5 cPoise±10%, 4.8 cPoise±10%, 6.0 cPoise±10%, 7.0 cPoise±10%, 7.1cPoise±10%, 7.2 cPoise±10%, 7.9 cPoise±10%, 8.9 cPoise±10%, 10.0cPoise±10%, 10.6 cPoise±10%, 11.4 cPoise±10%, 11.6 cPoise±10%, 11.8cPoise±10%, 12.4 cPoise±10%, 13.9 cPoise±10%, 14.0 cPoise±10%, 15.5cPoise±10%, or 17.9 cPoise±10%.

In one embodiment, the osmolality of the liquid pharmaceuticalformulation is between 100±15 mOsm/kg and 460±69 mOsm/kg. In oneembodiment, the osmolality of the liquid pharmaceutical formulation is103±15 mOsm/kg or about 103 mOsm/kg. In one embodiment, the osmolalityof the liquid pharmaceutical formulation is 195±29 mOsm/kg or about 195mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceuticalformulation is 220±33 mOsm/kg or about 220 mOsm/kg. In one embodiment,the osmolality of the liquid pharmaceutical formulation is 330±50mOsm/kg or about 330 mOsm/kg. In one embodiment, the osmolality of theliquid pharmaceutical formulation is 435±65 mOsm/kg or about 435mOsm/kg. In one embodiment, the osmolality of the liquid pharmaceuticalformulation is 440±66 mOsm/kg or about 440 mOsm/kg. In one embodiment,the osmolality of the liquid pharmaceutical formulation is 458±69mOsm/kg or about 458 mOsm/kg.

In one embodiment, at least 96% or at least 97% of the non-aggregatedand non-degraded form of the anti-PCSK9 antibody is recovered from theliquid pharmaceutical formulation after three months of storage of theliquid pharmaceutical formulation at −80° C., as determined by sizeexclusion chromatography. In one embodiment, at least 56% of thenon-basic and non-acidic form (i.e., main peak or main charge form) ofthe anti-PCSK9 antibody is recovered from the liquid pharmaceuticalformulation after three months of storage of the liquid pharmaceuticalformulation at −80° C., as determined by ion exchange chromatography.

In one embodiment, at least 96% or at least 97% of the non-aggregatedand non-degraded form of the anti-PCSK9 antibody is recovered from theliquid pharmaceutical formulation after three months of storage of theliquid pharmaceutical formulation at −30° C., as determined by sizeexclusion chromatography. In one embodiment, at least 56% of the maincharge form of the anti-PCSK9 antibody is recovered from the liquidpharmaceutical formulation after three months of storage of the liquidpharmaceutical formulation at −30° C., as determined by ion exchangechromatography.

In one embodiment, at least 96% or at least 97% of the non-aggregatedand non-degraded form of the anti-PCSK9 antibody is recovered from theliquid pharmaceutical formulation after three months of storage of theliquid pharmaceutical formulation at −20° C., as determined by sizeexclusion chromatography. In one embodiment, at least 56% of the maincharge form of the anti-PCSK9 antibody is recovered from the liquidpharmaceutical formulation after three months of storage of the liquidpharmaceutical formulation at −20° C., as determined by ion exchangechromatography.

In one embodiment, at least 96% of the non-aggregated and non-degradedform of the anti-PCSK9 antibody is recovered from the liquidpharmaceutical formulation after six months of storage of the liquidpharmaceutical formulation at 5° C., as determined by size exclusionchromatography. In one embodiment, at least 58% or 59% of the maincharge form of the anti-PCSK9 antibody is recovered from the liquidpharmaceutical formulation after three months of storage of the liquidpharmaceutical formulation at 5° C., as determined by ion exchangechromatography.

In one embodiment, at least 94% of the non-aggregated and non-degradedform of the anti-PCSK9 antibody is recovered from the liquidpharmaceutical formulation after six months of storage of the liquidpharmaceutical formulation at 25° C., as determined by size exclusionchromatography. In one embodiment, at least 45% or 47% of the non-basicand non-acidic form of the anti-PCSK9 antibody is recovered from theliquid pharmaceutical formulation after six months of storage of theliquid pharmaceutical formulation at 25° C., as determined by ionexchange chromatography.

In one embodiment, at least 91% or 92% of the non-aggregated andnon-degraded form of the anti-PCSK9 antibody is recovered from theliquid pharmaceutical formulation after 28 days of storage of the liquidpharmaceutical formulation at 45° C., as determined by size exclusionchromatography. In one embodiment, at least 35% or 37% of the non-basicand non-acidic form of the anti-PCSK9 antibody is recovered from theliquid pharmaceutical formulation after 28 days of storage of the liquidpharmaceutical formulation at 45° C., as determined by ion exchangechromatography.

In one aspect, a liquid pharmaceutical formulation is provided,comprising: (i) from 50±7.5 mg/ml to 175±26 mg/ml of a human antibodythat specifically binds to human PCSK9; (ii) from 0 mM to 40±6 mMhistidine; (iii) from 0% to 0.2%±0.03% (w/v) polysorbate 20; (iv) from0% to 12%±1.8% (w/v) sucrose; and (v) from 0 mM to 50±7.5 mM arginine,at a pH of from about 5.3 to about 6.7. The anti-PCSK9 antibody of thisaspect comprises a heavy chain variable region (HCVR) and a light chainvariable region (LCVR) such that the HCVR/LCVR combination comprisesheavy and light chain complementarity determining regions(HCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3), which comprise the amino acidsequences of SEQ ID NOs:2-3-4/SEQ ID NOs:6-7-8, respectively. In aparticular embodiment, the anti-PCSK9 antibody comprises a heavy chainvariable region (HCVR) and light chain variable region (LCVR) comprisingan amino acid sequence of SEQ ID NO:1 and SEQ ID NO:5, respectively(hereinafter “mAb-316P”).

In one embodiment of this aspect, the liquid formulation comprises (i)50±7.5 mg/mL of mAb-316P; (ii) 10±1.5 mM histidine; (iii) 0.1%±0.015%(w/v) polysorbate 20; and (iv) 6%±0.9% (w/v) sucrose, at a pH of6.0±0.3. In one embodiment of this particular formulation, the viscosityis about 1.7 cPoise. In one embodiment of this particular formulation,the osmolatlity is 220±44 mOsm/kg.

In another embodiment, the liquid formulation comprises (i) 100±20 mg/mLof mAb-316P; (ii) 20±4 mM histidine; (iii) 0.2%±0.04% (w/v) polysorbate20; and (iv) 12%±2.4% (w/v) sucrose, at a pH of 6.0±0.3. In oneembodiment of this particular formulation, the viscosity is about 3.5cPoise. In one embodiment of this particular formulation, theosmolatlity is 440±88 mOsm/kg.

In another embodiment, the liquid formulation comprises (i) 150±22.5mg/mL of mAb-316P; (ii) 10±1.5 mM histidine; (iii) 0.2%±0.03% or0.01%±0.0015% (w/v) polysorbate 20; and (iv) 10%±1.5% (w/v) sucrose, ata pH of 6.0±0.3. In one embodiment of this particular formulation, theviscosity is about 6 cPoise. In one embodiment of this particularformulation, the osmolatlity is 435±65.25 mOsm/kg. In one embodiment ofthis particular formulation, after storage of the formulation at 45° for28 days, ≧92% of the antibody is native and ≧35% of the antibody is ofthe main charge form. In one embodiment of this particular formulation,after storage of the formulation at 25° for six months, ≧94% of theantibody is native and ≧45% of the antibody is of the main charge form.In one embodiment of this particular formulation, after storage of theformulation at 5° for six months, ≧96% of the antibody is native and≧58% of the antibody is of the main charge form. In one embodiment ofthis particular formulation, after storage of the formulation at −20°for twelve months, ≧97% of the antibody is native and ≧56% of theantibody is of the main charge form. In one embodiment of thisparticular formulation, after storage of the formulation at −30° fortwelve months, ≧97% of the antibody is native and ≧56% of the antibodyis of the main charge form. In one embodiment of this particularformulation, after storage of the formulation at −80° for twelve months,≧97% of the antibody is native and ≧56% of the antibody is of the maincharge form.

In some embodiments of this particular formulation, ≧85% of the antibodyretains its biological potency after 28 days at 45° C., ≧82% after 28days at 37° C., and/or ≧98% after 28 days at 25° C. In some embodimentsof this particular formulation, ≧85% of the antibody retains itsbiological potency after six months at −20° C., ≧70% after six months at−30° C., and/or ≧79% after six months at −80° C. In some embodiments ofthis particular formulation, ≧81% of the antibody retains its biologicalpotency after eight freeze-thaw cycles, and/or ≧84% of the antibodyretains its biological activity after 120 minutes of agitation.

In another embodiment of this aspect, the liquid formulation comprises(i) 175±26.25 mg/mL of mAb-316P; (ii) 10±1.5 mM histidine; (iii)0.01%±0.0015% (w/v) polysorbate 20; (iv) 5%±0.75% (w/v) sucrose; and (v)50±7.5 mM arginine, at a pH of 6.0±0.3. In one embodiment of thisparticular formulation, the viscosity is about 10.6 cPoise. In oneembodiment of this particular formulation, the osmolatlity is 330±50mOsm/kg. In one embodiment of this particular formulation, after storageof the formulation at 45° for 28 days, ≧91% of the antibody is nativeand ≧38% of the antibody is of the main charge form. In one embodimentof this particular formulation, after storage of the formulation at 25°for six months, ≧94% of the antibody is native and ≧47% of the antibodyis of the main charge form. In one embodiment of this particularformulation, after storage of the formulation at 5° for six months, ≧96%of the antibody is native and ≧59% of the antibody is of the main chargeform. In one embodiment of this particular formulation, after storage ofthe formulation at −20° for three months, ≧96% of the antibody is nativeand ≧56% of the antibody is of the main charge form. In one embodimentof this particular formulation, after storage of the formulation at −30°for three months, ≧96% of the antibody is native and ≧56% of theantibody is of the main charge form. In one embodiment of thisparticular formulation, after storage of the formulation at −80° forthree months, ≧96% of the antibody is native and ≧56% of the antibody isof the main charge form.

In one aspect, a liquid pharmaceutical formulation of any of thepreceding aspects is provided in a container. In one embodiment, thecontainer is a polycarbonate vial. In another embodiment, the containeris a glass vial. In one embodiment, the glass vial is a type 1borosilicate glass vial with a fluorocarbon-coated butyl rubber stopper.In another embodiment, the container is a microinfuser. In anotherembodiment, the container is a syringe. In a specific embodiment, thesyringe comprises a fluorocarbon-coated plunger. In one specificembodiment, the syringe is a 1 mL long glass syringe containing lessthan about 500 parts per billion of tungsten equipped with a 27-Gneedle, a fluorocarbon-coated butyl rubber stopper, and a latex-free,non-cytotoxic rubber tip cap. In a more specific embodiment, the syringeis a Nuova Ompi 1 mL long glass syringe equipped with a 27-G thin wallneedle, a FluroTec®-coated 4023/50 rubber stopper, and a FM 27 rubbertip cap. In another specific embodiment, the syringe is a 1 mL or 3 mLplastic syringe fitted with a 27-G needle. In a more specificembodiment, the plastic syringe is distributed by Becton Dickinson.

In one aspect, a pharmaceutical formulation comprising (a) 175mg/mL±26.25 mg/mL of an anti-PCSK9 antibody, (b) 10 mM±1.5 mM histidine,pH 6±0.3, (c) 0.01% w/v±0.0015% polysorbate 20, (d) 5% w/v±0.75%sucrose, and (e) 50 mM±7.5 mM arginine, is provided, wherein (a) theantibody comprises an HCVD of SEQ ID NO:1 and an LCVD of SEQ ID NO:5,(b) over 90% of the antibodies in the formulation have a molecularweight of 155 kDa±1 kDa, (c) over 50% of the antibodies in theformulation have an isoelectric point of about 8.5, and (d) from 75% to90% of the antibodies in the formulation are fucosylated.

In one embodiment, the pharmaceutical formulation consists of (a) 175mg/mL±26.25 mg/mL of the anti-PCSK9 antibody of the immediatelypreceding paragraph, (b) 10 mM±1.5 mM histidine, pH 6±0.3, (c) 0.01%w/v±0.0015% polysorbate 20, (d) 5% w/v±0.75% sucrose, and (e) 50 mM±7.5mM arginine, in water.

In one aspect, a pharmaceutical formulation comprising (a) 150 mg/mLmL±22.5 mg/mL of an anti-PCSK9 antibody, (b) 10 mM±1.5 mM histidine, pH6±0.3, (c) 0.2% w/v±0.03% polysorbate 20, and (d) 10% w/v±1.5% sucroseis provided, wherein (i) the antibody comprises an HCVD of SEQ ID NO:1and an LCVD of SEQ ID NO:5, (ii) over 90% of the antibodies in theformulation have a molecular weight of 155 kDa±1 kDa, (iii) over 50% ofthe antibodies in the formulation have an isoelectric point of about8.5, and (iv) from 75% to 90% of the antibodies in the formulation arefucosylated.

In one embodiment, the pharmaceutical formulation consists of (a) 150mg/mL±22.5 mg/mL of the anti-PCSK9 antibody of the immediately precedingparagraph, (b) 10 mM±1.5 mM histidine, pH 6±0.3, (c) 0.2% w/v±0.03%polysorbate 20, and (d) 10% w/v±1.5% sucrose, in water.

In one aspect, a pharmaceutical formulation comprising (a) 150 mg/mLmL±22.5 mg/mL of an anti-PCSK9 antibody, (b) 10 mM±1.5 mM histidine, pH6±0.3, (c) 0.01% w/v±0.0015% polysorbate 20, and (d) 10% w/v±1.5%sucrose is provided, wherein (i) the antibody comprises an HCVD of SEQID NO:1 and an LCVD of SEQ ID NO:5, (ii) over 90% of the antibodies havea molecular weight of 155 kDa±1 kDa, (iii) over 50% of the antibodies inthe formulation have an isoelectric point of about 8.5, and (iv) from75% to 90% of the antibodies in the formulation are fucosylated.

In one embodiment, the pharmaceutical formulation consists of (a) 150mg/mL mL±22.5 mg/mL of the anti-PCSK9 antibody of the immediatelypreceding paragraph, (b) 10 mM±1.5 mM histidine, pH 6±0.3, (c) 0.01%w/v±0.0015% polysorbate 20, and (d) 10% w/v±1.5% sucrose, in water.

In one aspect, a pharmaceutical formulation comprising (a) 100 mg/mLmL±15 mg/mL of an anti-PCSK9 antibody, (b) 20 mM±3 mM histidine, pH6±0.3, (c) 0.2% w/v±0.03% polysorbate 20, and (d) 12% w/v±1.8% sucroseis provided, wherein (i) the antibody comprises an HCVD of SEQ ID NO:1and an LCVD of SEQ ID NO:5, (ii) over 90% of the antibodies in theformulation have a molecular weight of 155 kDa±1 kDa, (iii) over 50% ofthe antibodies in the formulation have an isoelectric point of about8.5, and (iv) from 75% to 90% of the antibodies in the formulation arefucosylated.

In one embodiment, the pharmaceutical formulation consists of (a) 100mg/mL mL±15 mg/mL of the anti-PCSK9 antibody of the immediatelypreceding paragraph, (b) 20 mM±3 mM histidine, pH 6±0.3, (c) 0.2%w/v±0.03% polysorbate 20, and (d) 12% w/v±1.8% sucrose, in water.

In one aspect, a pharmaceutical formulation comprising (a) 50 mg/mLmL±7.5 mg/mL of an antibody, (b) 10 mM±1.5 mM histidine, pH 6±0.3, (c)0.1% w/v±0.015% polysorbate 20, and (d) 6% w/v±0.9% sucrose is provided,wherein (i) the antibody comprises an HCVD of SEQ ID NO:1 and an LCVD ofSEQ ID NO:5, (ii) over 90% of the antibodies in the formulation have amolecular weight of 155 kDa±1 kDa, (iii) over 50% of the antibodies inthe formulation have an isoelectric point of about 8.5, and (iv) from75% to 90% of the antibodies in the formulation are fucosylated.

In one embodiment, the pharmaceutical formulation consists of (a) 50mg/mL mL±7.5 mg/mL of the anti-PCSK9 antibody of the immediatelypreceding paragraph, (b) 10 mM±1.5 mM histidine, pH 6±0.3, (c) 0.1%w/v±0.015% polysorbate 20, and (d) 6% w/v±0.9% sucrose, in water.

In one aspect, a method is provided for preparing a freeze-driedcomposition that comprises an anti-PCSK9 antibody and less than 0.3%water. The method comprises the steps of (a) combining in a glass vialwater, an anti-PCSK9 antibody, histidine, sucrose, and polysorbate 20,(b) then holding the combination at about 5° C. for about 60 minutes,(c) then decreasing the temperature at a rate of about 0.5° C. perminute, (d) then holding the combination at about −45° C. for about 120minutes, (e) then reducing the atmospheric pressure to about 100 mTorr,(f) then increasing the temperature at a rate of about 0.5° C. perminute, (g) then holding the combination at about −25° C. for about 78hours, (h) then increasing the temperature at a rate of 0.2° C. perminute, (i) then holding the combination at about 35° C. for about 6hours, (j) then decreasing the temperature at a rate of about 0.5° C.,(k) and then holding the combination at about 25° for about 60 minutes,prior to storage.

In one embodiment, the method further comprises the steps of (l)backfilling the glass vial containing the combination of step (k) withnitrogen gas, and (m) stoppering the vial under about 80% of atmosphericpressure. In one embodiment, the composition is brought to 2-8° C. afterstep (i), (j) or (k) and prior to the step of stoppering the vial.

In some embodiments, at step (a) the anti-PCSK9 antibody is at 50mg/mL±7.5 mg/mL, the histidine is at 10 mM±1.5 mM (pH 6.0), thepolysorbate 20 is at 0.1%±0.015%, and the sucrose is at 6%±0.9%. In oneembodiment, the anti-PCSK9 antibody comprises an HCDR1 of SEQ ID NO:2,an HCDR2 of SEQ ID NO:3, an HCDR3 of SEQ ID NO:4, an LCDR1 of SEQ ID NO:6, an LCDR2 of SEQ ID NO:7, and an LCDR3 of SEQ ID NO:8.mAb-316P. In oneembodiment, the anti-PCSK9 antibody comprises an HCVD of SEQ ID NO:1 andan LCVD of SEQ ID NO:5. In one embodiment, (i) the antibody comprises anHCVD of SEQ ID NO:1 and an LCVD of SEQ ID NO:5, (ii) over 90% of theantibodies in the combination have a molecular weight of 155 kDa±1 kDa,(iii) over 50% of the antibodies in the combination have an isoelectricpoint of about 8.5, and (iv) from 75% to 90% of the antibodies in thecombination are fucosylated.

In one aspect, a freeze-dried pharmaceutical composition comprising ananti-PCSK9 antibody and less than 0.3% water, which is producedaccording to the method of the preceding aspect, is provided.

In one aspect, a pharmaceutical composition is provided, which comprisesthe freeze-dried pharmaceutical composition of the preceding aspectresuspended in water. In one embodiment, the pharmaceutical compositionconsists of 50 mg/mL±7.5 mg/mL of the anti-PCSK9 antibody, 10 mM±1.5 mMhistidine (pH 6.0), 0.1%±0.015% polysorbate 20, and 6%±0.9% sucrose, inwater. In one embodiment, the pharmaceutical composition consists of 100mg/mL±15 mg/mL of the anti-PCSK9 antibody, 20 mM±3 mM histidine (pH6.0), 0.2%±0.03% polysorbate 20, and 12%±1.8% sucrose, in water. Inanother embodiment, the pharmaceutical composition consists of 150mg/mL±22.5 mg/mL of the anti-PCSK9 antibody, 30 mM±4.5 mM histidine (pH6.0), 0.3%±0.045% polysorbate 20, and 18%±2.7% sucrose, in water. In yetanother embodiment, the pharmaceutical composition consists of 175mg/mL±26.25 mg/mL of the anti-PCSK9 antibody, 35 mM±5.25 mM histidine(pH 6.0), 0.35%±0.0525% polysorbate 20, and 21%±3.15% sucrose, in water.

In some embodiments, the anti-PCSK9 antibody comprises an HCVD of SEQ IDNO:1 and an LCVD of SEQ ID NO:5, and (b) over 90% of the antibodies inthe composition have a molecular weight of 155 kDa±1 kDa, (c) over 50%of the antibodies in the composition have an isoelectric point of about8.5, and (d) from 75% to 90% of the antibodies in the composition arefucosylated.

In one aspect, a pharmaceutical composition of any one of the precedingaspects is provided, wherein said composition is contained in acontainer. In one embodiment, the container is a vial, which in someembodiments is a glass vial. In another embodiment, the container is asyringe. In some embodiments, the syringe is a low-tungsten glasssyringe. In one embodiment, the syringe is a Nuova Ompi 1 mL long glasssyringe equipped with a 27-G thin wall needle, a FluroTec®-coated4023/50 rubber stopper, and a FM 27 rubber tip cap.

In one aspect, a kit comprising a pharmaceutical composition of any oneof the preceding aspects, a container, and instructions is provided. Inone embodiment, the container is a prefilled syringe. In a particularembodiment, the syringe is a Nuova Ompi 1 mL long glass syringe equippedwith a 27-G thin wall needle, a FluroTec®-coated 4023/50 rubber stopper,and a FM 27 rubber tip cap. Other embodiments of the present inventionwill become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about”, when used in reference to a particular recited numerical valueor range of values, means that the value may vary from the recited valueby no more than 1%. For example, as used herein, the expression “about100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2,99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference todescribe in their entirety.

Pharmaceutical Formulations

As used herein, the expression “pharmaceutical formulation” means acombination of at least one active ingredient (e.g., a small molecule,macromolecule, compound, etc. which is capable of exerting a biologicaleffect in a human or non-human animal), and at least one inactiveingredient which, when combined with the active ingredient or one ormore additional inactive ingredients, is suitable for therapeuticadministration to a human or non-human animal. The term “formulation”,as used herein, means “pharmaceutical formulation” unless specificallyindicated otherwise. The present invention provides pharmaceuticalformulations comprising at least one therapeutic polypeptide. Accordingto certain embodiments of the present invention, the therapeuticpolypeptide is an antibody, or an antigen-binding fragment thereof,which binds specifically to human proprotein convertase subtilisin/kexintype 9 (PCSK9) protein. More specifically, the present inventionincludes pharmaceutical formulations that comprise: (i) a human antibodythat specifically binds to human PCSK9 (ii) a histidine buffer; (iii) anorganic cosolvent that is a non-ionic surfactant; (iv) thermalstabilizer that is a carbohydrate; and, optionally, (v) a viscosityreducer that is a salt. Specific exemplary components and formulationsincluded within the present invention are described in detail below.

Antibodies that Bind Specifically to PCSK9

The pharmaceutical formulations of the present invention may comprise ahuman antibody, or an antigen-binding fragment thereof, that bindsspecifically to human PCSK9. As used herein, the term “PCSK9” means ahuman proprotein convertase belonging to the proteinase K subfamilty ofthe secretory subtilase family. Evidence suggests that PCSK9 increasesplasma LDL levels by binding to the low-density lipoprotein particlereceptor and promoting its degradation. An exemplary human PCSK9 aminoacid sequence is described in SEQ ID NO:9. Antibodies to human PCSK9 aredescribed in patent application publications US 2010/0166768, US2011/0065902, and WO 2010/077854.

The term “antibody”, as used herein, is generally intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM); however, immunoglobulinmolecules consisting of only heavy chains (i.e., lacking light chains)are also encompassed within the definition of the term “antibody”. Eachheavy chain comprises a heavy chain variable region (abbreviated hereinas HCVR or V_(H)) and a heavy chain constant region. The heavy chainconstant region comprises three domains, CH1, CH2 and CH3. Each lightchain comprises a light chain variable region (abbreviated herein asLCVR or V_(L)) and a light chain constant region. The light chainconstant region comprises one domain (CL1). The V_(H) and V_(L) regionscan be further subdivided into regions of hypervariability, termedcomplementary determining regions (CDRs), interspersed with regions thatare more conserved, termed framework regions (FR). Each V_(H) and V_(L)is composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

Unless specifically indicated otherwise, the term “antibody”, as usedherein, shall be understood to encompass complete antibody molecules aswell as antigen-binding fragments thereof. The term “antigen-bindingportion” or “antigen-binding fragment” of an antibody (or simply“antibody portion” or “antibody fragment”), as used herein, refers toone or more fragments of an antibody that retain the ability tospecifically bind to human PCSK9 or an epitope thereof.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds human PCSK9 is substantially free of antibodies that specificallybind antigens other than human PCSK9).

The term “specifically binds”, or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by a dissociation constant of at least about 1×10⁻⁶ M orgreater. Methods for determining whether two molecules specifically bindare well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds human PCSK9 may, however, have cross-reactivityto other antigens, such as PCSK9 molecules from other species(orthologs). In the context of the present invention, multispecific(e.g., bispecific) antibodies that bind to human PCSK9 as well as one ormore additional antigens are deemed to “specifically bind” human PCSK9.Moreover, an isolated antibody may be substantially free of othercellular material or chemicals.

Exemplary anti-human PCSK9 antibodies that may be included in thepharmaceutical formulations of the present invention are set forth inpatent application publications US 2010/0166768, US 2011/0065902, and WO2010/077854, the disclosures of which are incorporated by reference intheir entirety.

According to certain embodiments of the present invention, theanti-human PCSK9 mAb-316P antibody is a human IgG1 comprising a heavychain variable region that is of the IGHV3-23 subtype and a light chainvariable region that is of the IGKV4-1 subtype (see Barbie and Lefranc,The Human Immunoglobulin Kappa Variable (IGKV) Genes and Joining (IGKJ)Segments, Exp. Clin. Immunogenet. 1998; 15:171-183; and Scaviner, D. etal., Protein Displays of the Human Immunoglobulin Heavy, Kappa andLambda Variable and Joining Regions, Exp. Clin. Immunogenet., 1999;16:234-240).

In some embodiments, the anti-human PCSK9 mAb-316P comprises at leastone amino acid substitution, which results in a charge change at anexposed surface of the antibody relative to the germline IGKV4-1sequence. The germline IGKV4-1 sequence, and the amino acid positionassignment numbers presented herein comport with the internationalImmunogenetics (IMGT) information system, as described in Lefranc,M.-P., et al., IMGT®, the international ImMunoGeneTics informationSystem®, Nucl. Acids Res, 37, D1006-D1012 (2009).

In some embodiments, the exposed surface comprises a complementaritydetermining region (CDR). In some embodiments, the amino acidsubstitution or substitutions are selected from the group consisting ofa basic amino acid substituted for an uncharged polar amino acid withinCDR1 (e.g., at position 32) of IGKV4-1. Unique permutations in thecharge distribution of an antibody, especially at an environmentalinterface (such as, e.g., in a CDR) would be expected to createunpredictable conditions for maintaining or advancing the stability ofthe antibody in solution.

In some embodiments, the anti-human PCSK9 mAb-316P antibody comprises atleast one amino acid substitution, which creates a charge change withina framework region of a variable region of the antibody relative to thegermline IGHV3-23 sequence or the germline IGKV4-1 sequence. In someembodiments, the amino acid substitution or substitutions are selectedfrom the group consisting of (a) a hydrophobic amino acid substitutedfor a polar amino acid in framework region 3 (FR3) (e.g., at position77) of IGHV3-23, and (b) a polar amino acid substituted for a basicamino acid in framework region 2 (FR2) (e.g., at position 51) ofIGKV4-1. Changes in the ability of the peptide chain to fold, especiallywithin a framework region, which affects the CDR interface with thesolvent, would be expected to create unpredictable conditions formaintaining or advancing the stability of the antibody in solution.

According to certain embodiments of the present invention, theanti-human PCSK9 antibody, or antigen-binding fragment thereof,comprises a heavy chain complementary determining region (HCDR) 1 of SEQID NO: 2, an HCDR2 of SEQ ID NO:3, and an HCDR3 of SEQ ID NO: 4. Incertain embodiments, the anti-human PCSK9 antibody, or antigen-bindingfragment thereof, comprises an HCVD of SEQ ID NO:1.

According to certain embodiments of the present invention, theanti-human PCSK9, or antigen-binding fragment thereof, comprises a light(kappa) chain complementary determining region (LCDR) 1 of SEQ ID NO: 6,an LCDR2 of SEQ ID NO: 7, and an LCDR3 of SEQ ID NO: 8. In certainembodiments, the anti-human PCSK9 antibody, or antigen-binding fragmentthereof, comprises an LCVD of SEQ ID NO:5.

The non-limiting, exemplary antibody used in the Examples herein isreferred to as “mAb-316P”. This antibody is also referred to in U.S.Pat. No. 7,608,693 as H4H098P. mAb-316P (H4H098P) comprises an HCVR/LCVRamino acid sequence pair having SEQ ID NOs:1/5, andHCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3 domains represented by SEQ IDNOs:2-3-4/SEQ ID NOs:6-7-8.

The amount of antibody, or antigen-binding fragment thereof, containedwithin the pharmaceutical formulations of the present invention may varydepending on the specific properties desired of the formulations, aswell as the particular circumstances and purposes for which theformulations are intended to be used. In certain embodiments, thepharmaceutical formulations are liquid formulations that may contain50±7.5 mg/mL to 250±37.5 mg/mL of antibody; 60±9 mg/mL to 240±36 mg/mLof antibody; 70±10.5 mg/mL to 230±34.5 mg/mL of antibody; 80±12 mg/mL to220±33 mg/mL of antibody; 90±13.5 mg/mL to 210±31.5 mg/mL of antibody;100±15 mg/mL to 200±30 mg/mL of antibody; 110±16.5 mg/mL to 190±28.5mg/mL of antibody; 120±18 mg/mL to 180±27 mg/mL of antibody; 130±19.5mg/mL to 170±25.5 mg/mL of antibody; 140±21 mg/mL to 160±24 mg/mL ofantibody; 150±22.5 mg/mL of antibody; or 175±26.25 mg/ml. For example,the formulations of the present invention may comprise about 50 mg/mL;about 60 mg/mL; about 65 mg/mL; about 70 mg/mL; about 75 mg/mL; about 80mg/mL; about 85 mg/mL; about 90 mg/mL; about 95 mg/mL; about 100 mg/mL;about 105 mg/mL; about 110 mg/mL; about 115 mg/mL; about 120 mg/mL;about 125 mg/mL; about 130 mg/mL; about 135 mg/mL; about 140 mg/mL;about 145 mg/mL; about 150 mg/mL; about 155 mg/mL; about 160 mg/mL;about 165 mg/mL; about 170 mg/mL; about 175 mg/mL; about 180 mg/mL;about 185 mg/mL; about 190 mg/mL; about 195 mg/mL; about 200 mg/mL;about 205 mg/mL; about 210 mg/mL; about 215 mg/mL; about 220 mg/mL;about 225 mg/mL; about 230 mg/mL; about 235 mg/mL; about 240 mg/mL;about 245 mg/mL; or about 250 mg/mL of an antibody or an antigen-bindingfragment thereof, that binds specifically to human PCSK9.

Excipients and pH

The pharmaceutical formulations of the present invention comprise one ormore excipients. The term “excipient”, as used herein, means anynon-therapeutic agent added to the formulation to provide a desiredconsistency, viscosity or stabilizing effect.

In certain embodiments, the pharmaceutical formulation of the inventioncomprises at least one organic cosolvent in a type and in an amount thatstabilizes the human PCSK9 antibody under conditions of rough handlingor agitation, such as, e.g., vortexing. In some embodiments, what ismeant by “stabilizes” is the prevention of the formation of more than 3%aggregated antibody of the total amount of antibody (on a molar basis)over the course of rough handling. In some embodiments, rough handlingis vortexing a solution containing the antibody and the organiccosolvent for about 60 minutes or about 120 minutes.

In certain embodiments, the organic cosolvent is a non-ionic surfactant,such as an alkyl poly(ethylene oxide). Specific non-ionic surfactantsthat can be included in the formulations of the present inventioninclude, e.g., polysorbates such as polysorbate 20, polysorbate 28,polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80,polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 181,poloxamer 188, poloxamer 407; or polyethylene glycol (PEG). Polysorbate20 is also known as TWEEN® 20 (polysorbate 20), sorbitan monolaurate andpolyoxyethylenesorbitan monolaurate. Poloxamer 188 is also known asPluronic® F-68.

The amount of non-ionic surfactant contained within the pharmaceuticalformulations of the present invention may vary depending on the specificproperties desired of the formulations, as well as the particularcircumstances and purposes for which the formulations are intended to beused. In certain embodiments, the formulations may contain 0.01%±0.0015%to 0.2%±0.03% surfactant. For example, the formulations of the presentinvention may comprise about 0.0085%; about 0.01%; about 0.02%; about0.03%; about 0.04%; about 0.05%; about 0.06%; about 0.07%; about 0.08%;about 0.09%; about 0.1%; about 0.11%; about 0.12%; about 0.13%; about0.14%; about 0.15%; about 0.16%; about 0.17%; about 0.18%; about 0.19%;about 0.20%; about 0.21%; about 0.22%; or about 0.23% polysorbate 20 orpoloxamer 188.

The pharmaceutical formulations of the present invention may alsocomprise one or more stabilizers in a type and in an amount thatstabilizes the human PCSK9 antibody under conditions of thermal stress.In some embodiments, what is meant by “stabilizes” is maintaininggreater than about 91% of the antibody in a native conformation when thesolution containing the antibody and the thermal stabilizer is kept atabout 45° C. for up to about 28 days. In some embodiments, what is meantby “stabilizes” is wherein less than about 6% of the antibody isaggregated when the solution containing the antibody and the thermalstabilizer is kept at about 45° C. for up to about 28 days. As usedherein, “native” means the major form of the antibody by size exclusion,which is generally an intact monomer of the antibody.

In certain embodiments, the thermal stabilizer is a sugar or sugaralcohol selected from sucrose, trehalose and mannitol, or anycombination thereof, the amount of which contained within theformulation can vary depending on the specific circumstances andintended purposes for which the formulation is used. In certainembodiments, the formulations may contain about 3% to about 14% sugar orsugar alcohol; about 4% to about 13% sugar or sugar alcohol; about 5% toabout 12% sugar or sugar alcohol; about 6% to about 11% sugar or sugaralcohol; about 7% to about 10% sugar or sugar alcohol; about 8% to about9% sugar or sugar alcohol; about 4% to about 6% sugar or sugar alcohol;about 5% to about 7% sugar or sugar alcohol; about 9% to about 11% sugaror sugar alcohol; or about 11% to about 13% sugar or sugar alcohol. Forexample, the pharmaceutical formulations of the present invention maycomprise 4%±0.6%; 5%±0.75%; 6%±0.9%; 7%±1.05%; 8%±1.2%; 9%±1.35%;10%±1.5%; 11%±1.65%; 12%±1.8%; 13%±1.95%; or about 14%±2.1% sugar orsugar alcohol (e.g., sucrose, trehalose or mannitol).

The pharmaceutical formulations of the present invention may alsocomprise a buffer or buffer system, which serves to maintain a stable pHand to help stabilize the human PCSK9 antibody. In some embodiments,what is meant by “stabilizes” is wherein less than 4.5%±0.5% or lessthan 6.0±0.5% of the antibody is aggregated when the solution containingthe antibody and the buffer is kept at about 45° C. for up to about 28days. In some embodiments, what is meant by “stabilizes” is wherein lessthan 3%±0.5% or less than 2.6%±0.5% of the antibody is aggregated whenthe solution containing the antibody and the buffer is kept at about 37°C. for up to about 28 days. In some embodiments, what is meant by“stabilizes” is wherein at least 91%±0.5% or at least 92%±0.5% of theantibody is in its native conformation as determined by size exclusionchromatography when the solution containing the antibody and the bufferis kept at about 45° C. for up to about 28 days. In some embodiments,what is meant by “stabilizes” is wherein at least 94%±0.5% or at least95%±0.5% of the antibody is in its native conformation as determined bysize exclusion chromatography when the solution containing the antibodyand the buffer is kept at about 37° C. for up to about 28 days. By“native” or “native conformation”, what is meant is the antibodyfraction that is not aggregated or degraded. This is generallydetermined by an assay that measures the relative size of the antibodyentity, such as a size exclusion chromatographic assay. Thenon-aggregated and non-degraded antibody elutes at a fraction thatequates to the native antibody, and is generally the main elutionfraction. Aggregated antibody elutes at a fraction that indicates a sizegreater than the native antibody. Degraded antibody elutes at a fractionthat indicates a size less than the native antibody.

In some embodiments, what is meant by “stabilizes” is wherein at least38%±0.5% or at least 29%±0.5% of the antibody is in its main charge formas determined by cation exchange chromatography when the solutioncontaining the antibody and the buffer is kept at about 45° C. for up toabout 28 days. In some embodiments, what is meant by “stabilizes” iswherein at least 46%±0.5% or at least 39%±0.5% of the antibody is in itsmain charge form as determined by cation exchange chromatography whenthe solution containing the antibody and the buffer is kept at about 37°C. for up to about 28 days. By “main charge” or “main charge form”, whatis meant is the fraction of antibody that elutes from an ion exchangeresin in the main peak, which is generally flanked by more “basic” peakson one side and more “acidic” peaks on the other side.

The pharmaceutical formulations of the present invention may have a pHof from about 5.2 to about 6.4. For example, the formulations of thepresent invention may have a pH of about 5.5; about 5.6; about 5.7;about 5.8; about 5.9; about 6.0; about 6.1; about 6.2; about 6.3; about6.4; or about 6.5. In some embodiments, the pH is 6.0±0.4; 6.0±0.3;6.0±0.2; 6.0±0.1; about 6.0; or 6.0.

In some embodiments, the buffer or buffer system comprises at least onebuffer that has a buffering range that overlaps fully or in part therange of pH 5.5-7.4. In one embodiment, the buffer has a pKa of about6.0±0.5. In certain embodiments, the buffer comprises a histidinebuffer. In certain embodiments, the histidine is present at aconcentration of 5 mM±0.75 mM to 15 mM±2.25 mM; 6 mM±0.9 mM to 14 mM±2.1mM; 7 mM±1.05 mM to 13 mM±1.95 mM; 8 mM±1.2 mM to 12 mM±1.8 mM; 9mM±1.35 mM to 11 mM±1.65 mM; 10 mM±1.5 mM; or about 10 mM. In certainembodiments, the buffer system comprises histidine at 10 mM±1.5 mM, at apH of 6.0±0.3.

The pharmaceutical formulations of the present invention may alsocomprise one or more excipients that serve to maintain a reducedviscosity or to lower the viscosity of formulations containing a highconcentration of anti-PCSK9 antibody drug substance (e.g.,generally >150 mg/ml of antibody). In some embodiments, the formulationcomprises arginine in an amount sufficient to maintain the viscosity ofthe liquid formulation at less than 20±3 cPoise, less than 15±2.25cPoise, or less than 11±1.65 cPoise. In some embodiments, theformulation comprises arginine in an amount sufficient to maintain theviscosity at or below 10.6±1.59 cPoise. In certain embodiments, thepharmaceutical formulation of the present invention contains arginine,preferably as L-arginine hydrochloride, at a concentration of 10 mM±1.5mM to 90 mM±13.5 mM, 20 mM±3 mM to 80 mM±12 mM, 30 mM±4.5 mM to 70mM±10.5, 40 mM±6 mM to 60±9 mM or 50 mM±7.5 mM.

Exemplary Formulations

According to one aspect of the present invention, the pharmaceuticalformulation is a low viscosity, generally physiologically isotonicliquid formulation, which comprises: (i) a human antibody thatspecifically binds to human PCSK9 (e.g., mAb-316P), at a concentrationof 50 mg/mL±7.5 mg/mL, 100 mg/ml±15 mgmL, 150 mg/mL±22.5 mg/mL, or 175mg/mL±26.25 mg/mL; (ii) a buffer system that provides sufficientbuffering at about pH 6.0±0.3; (iii) a sugar which serves inter alia asa thermal stabilizer; (iv) an organic cosolvent, which protects thestructural integrity if the antibody; and (v) a salt of an amino acid,which serves to keep the viscosity manageable for injection in aconvenient volume for subcutaneous administration.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9 andwhich comprises a substituted IGHV3-23 type heavy chain variable regionand a substituted IGLV4-1 type light chain variable region (e.g.,mAb-316P) at a concentration from 50±7.5 mg/mL to about 175±26.25 mg/mL;(ii) a buffer system comprising histidine, which buffers effectively atabout pH 6.0±0.3; (iii) sucrose; (iv) a non-ionic detergent, such as apolysorbate; and optionally (v) an arginine salt.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9, andwhich comprises an HCDR1 of SEQ ID NO:2, an HCDR2 of SEQ ID NO:3, anHCDR3 of SEQ ID NO:4, an LCDR1 of SEQ ID NO:6, an LCDR2 of SEQ ID NO:7,and an LCDR3 of SEQ ID NO:8, at a concentration of 175 mg/ml±26.25mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers at pH 6.0±0.3;(iii) sucrose at 5% w/v±0.75% w/v; (iv) polysorbate 20 at 0.01%w/v±0.0015% w/v; and (v) L-arginine hydrochloride at 50 mM±7.5 mM.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9, andwhich comprises an HCDR1 of SEQ ID NO:2, an HCDR2 of SEQ ID NO:3, anHCDR3 of SEQ ID NO:4, an LCDR1 of SEQ ID NO:6, an LCDR2 of SEQ ID NO:7,and an LCDR3 of SEQ ID NO:8, at a concentration of about 150 mg/ml±22.5mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers at pH 6.0±0.3;(iii) sucrose at 10% w/v±1.5% w/v; and (iv) polysorbate 20 at 0.2%w/v±0.03% w/v or 0.01% w/v±0.0015% w/v.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9, andwhich comprises an HCDR1 of SEQ ID NO:2, an HCDR2 of SEQ ID NO:3, anHCDR3 of SEQ ID NO:4, an LCDR1 of SEQ ID NO:6, an LCDR2 of SEQ ID NO:7,and an LCDR3 of SEQ ID NO:8, at a concentration of about 100 mg/mL±15mg/mL; (ii) histidine at about 20 mM±3 mM, which buffers at pH 6.0±0.3;(iii) sucrose at 12% w/v±1.8% w/v; and (iv) polysorbate 20 at 0.2%w/v±0.03% w/v or 0.01% w/v±0.0015% w/v.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9, andwhich comprises an HCDR1 of SEQ ID NO:2, an HCDR2 of SEQ ID NO:3, anHCDR3 of SEQ ID NO:4, an LCDR1 of SEQ ID NO:6, an LCDR2 of SEQ ID NO:7,and an LCDR3 of SEQ ID NO:8, at a concentration of about 50 mg/mL±7.5mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers at pH 6.0±0.3;(iii) sucrose at 6% w/v±0.9% w/v; and (iv) polysorbate 20 at 0.1%w/v±0.015% w/v or 0.01% w/v±0.0015% w/v.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9, andwhich comprises a heavy chain variable domain of SEQ ID NO:1, and alight chain variable domain of SEQ ID NO:5, at a concentration of 175mg/ml±26.25 mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers at pH6.0±0.3; (iii) sucrose at 5% w/v±0.75% w/v; (iv) polysorbate 20 at 0.01%w/v±0.0015% w/v; and (v) L-arginine hydrochloride at 50 mM±7.5 mM.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9, andwhich comprises a heavy chain variable domain of SEQ ID NO:1, and alight chain variable domain of SEQ ID NO:5, at a concentration of about150 mg/ml±22.5 mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers atpH 6.0±0.3; (iii) sucrose at 10% w/v±1.5% w/v; and (iv) polysorbate 20at 0.2% w/v±0.03% w/v or 0.01% w/v±0.0015% w/v.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9, andwhich comprises a heavy chain variable domain of SEQ ID NO:1, and alight chain variable domain of SEQ ID NO:5, at a concentration of about100 mg/mL±15 mg/mL; (ii) histidine at about 20 mM±3 mM, which buffers atpH 6.0±0.3; (iii) sucrose at 12% w/v±1.8% w/v; and (iv) polysorbate 20at 0.2% w/v±0.03% w/v or 0.01% w/v±0.0015% w/v.

According to one embodiment, the pharmaceutical formulation comprises:(i) a human IgG1 antibody that specifically binds to human PCSK9, andwhich comprises a heavy chain variable domain of SEQ ID NO:1, and alight chain variable domain of SEQ ID NO:5, at a concentration of about50 mg/mL±7.5 mg/mL; (ii) histidine at 10 mM±1.5 mM, which buffers at pH6.0±0.3; (iii) sucrose at 6% w/v±0.9% w/v; and (iv) polysorbate 20 at0.1% w/v±0.015% w/v or 0.01% w/v±0.0015% w/v.

Additional non-limiting examples of pharmaceutical formulationsencompassed by the present invention are set forth elsewhere herein,including the working Examples presented below.

Stability and Viscosity of the Pharmaceutical Formulations

The pharmaceutical formulations of the present invention typicallyexhibit high levels of stability. The term “stable”, as used herein inreference to the pharmaceutical formulations, means that the antibodieswithin the pharmaceutical formulations retain an acceptable degree ofchemical structure or biological function after storage under definedconditions. A formulation may be stable even though the antibodycontained therein does not maintain 100% of its chemical structure orbiological function after storage for a defined amount of time. Undercertain circumstances, maintenance of about 90%, about 95%, about 96%,about 97%, about 98% or about 99% of an antibody's structure or functionafter storage for a defined amount of time may be regarded as “stable”.

Stability can be measured, inter alia, by determining the percentage ofnative antibody that remains in the formulation after storage for adefined amount of time at a defined temperature. The percentage ofnative antibody can be determined by, inter alia, size exclusionchromatography (e.g., size exclusion high performance liquidchromatography [SE-HPLC]), such that native means non-aggregated andnon-degraded. An “acceptable degree of stability”, as that phrase isused herein, means that at least 90% of the native form of the antibodycan be detected in the formulation after storage for a defined amount oftime at a given temperature. In certain embodiments, at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native formof the antibody can be detected in the formulation after storage for adefined amount of time at a defined temperature. The defined amount oftime after which stability is measured can be at least 14 days, at least28 days, at least 1 month, at least 2 months, at least 3 months, atleast 4 months, at least 5 months, at least 6 months, at least 7 months,at least 8 months, at least 9 months, at least 10 months, at least 11months, at least 12 months, at least 18 months, at least 24 months, ormore. The defined temperature at which the pharmaceutical formulationmay be stored when assessing stability can be any temperature from about−80° C. to about 45° C., e.g., storage at about −80° C., about −30° C.,about −20° C., about 0° C., about 4°-8° C., about 5° C., about 25° C.,about 35° C., about 37° C., or about 45° C. For example, apharmaceutical formulation may be deemed stable if after 6 months ofstorage at 5° C., greater than about 95%, 96%, 97% or 98% of nativeantibody is detected by SE-HPLC. A pharmaceutical formulation may alsobe deemed stable if after 6 months of storage at 25° C., greater thanabout 94%, 95%, 96%, 97% or 98% of native antibody is detected bySE-HPLC. A pharmaceutical formulation may also be deemed stable if after28 days of storage at 45° C., greater than about 91%, 92%, 93%, 94%,95%, 96%, 97% or 98% of native antibody is detected by SE-HPLC. Apharmaceutical formulation may also be deemed stable if after threemonths of storage at −20° C., greater than about 96%, 97%, or 98% ofnative antibody is detected by SE-HPLC. A pharmaceutical formulation mayalso be deemed stable if after three months of storage at −30° C.,greater than about 96%, 97% or 98% of native antibody is detected bySE-HPLC. A pharmaceutical formulation may also be deemed stable if afterthree months of storage at −80° C., greater than about 96%, 97% or 98%of native antibody is detected by SE-HPLC.

Stability can be measured, inter alia, by determining the percentage ofantibody that forms in an aggregate within the formulation after storagefor a defined amount of time at a defined temperature, wherein stabilityis inversely proportional to the percent aggregate that is formed. Thepercentage of aggregated antibody can be determined by, inter alia, sizeexclusion chromatography (e.g., size exclusion high performance liquidchromatography [SE-HPLC]). An “acceptable degree of stability”, as thatphrase is used herein, means that at most 6% of the antibody is in anaggregated form detected in the formulation after storage for a definedamount of time at a given temperature. In certain embodiments anacceptable degree of stability means that at most about 6%, 5%_(,)4%_(,) 3%_(,) 2%_(,) 1%, 0.5%, or 0.1% of the antibody can be detectedin an aggregate in the formulation after storage for a defined amount oftime at a given temperature. The defined amount of time after whichstability is measured can be at least 2 weeks, at least 28 days, atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months, at least 7 months, at least 8months, at least 9 months, at least 10 months, at least 11 months, atleast 12 months, at least 18 months, at least 24 months, or more. Thetemperature at which the pharmaceutical formulation may be stored whenassessing stability can be any temperature from about −80° C. to about45° C., e.g., storage at about −80° C., about −30° C., about −20° C.,about 0° C., about 4°-8° C., about 5° C., about 25° C., about 35° C.,about 37° C. or about 45° C. For example, a pharmaceutical formulationmay be deemed stable if after six months of storage at 5° C., less thanabout 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detected in anaggregated form. A pharmaceutical formulation may also be deemed stableif after six months of storage at 25° C., less than about 4%, 3%, 2%,1%, 0.5%, or 0.1% of the antibody is detected in an aggregated form. Apharmaceutical formulation may also be deemed stable if after 28 days ofstorage at 45° C., less than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%of the antibody is detected in an aggregated form. A pharmaceuticalformulation may also be deemed stable if after three months of storageat −20° C., −30° C., or −80° C. less than about 3%, 2%, 1%, 0.5%, or0.1% of the antibody is detected in an aggregated form.

Stability can be measured, inter alia, by determining the percentage ofantibody that migrates in a more acidic fraction during ion exchange(“acidic form”) than in the main fraction of antibody (“main chargeform”), wherein stability is inversely proportional to the fraction ofantibody in the acidic form. While not wishing to be bound by theory,deamidation of the antibody may cause the antibody to become morenegatively charged and thus more acidic relative to the non-deamidatedantibody (see, e.g., Robinson, N., Protein Deamidation, PNAS, Apr. 16,2002, 99(8):5283-5288). The percentage of “acidified” antibody can bedetermined by, inter alia, ion exchange chromatography (e.g., cationexchange high performance liquid chromatography [CEX-HPLC]). An“acceptable degree of stability”, as that phrase is used herein, meansthat at most 49% of the antibody is in a more acidic form detected inthe formulation after storage for a defined amount of time at a definedtemperature. In certain embodiments an acceptable degree of stabilitymeans that at most about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody can be detected in anacidic form in the formulation after storage for a defined amount oftime at a given temperature. The defined amount of time after whichstability is measured can be at least 2 weeks, at least 28 days, atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months, at least 7 months, at least 8months, at least 9 months, at least 10 months, at least 11 months, atleast 12 months, at least 18 months, at least 24 months, or more. Thetemperature at which the pharmaceutical formulation may be stored whenassessing stability can be any temperature from about −80° C. to about45° C., e.g., storage at about −80° C., about −30° C., about −20° C.,about 0° C., about 4°-8° C., about 5° C., about 25° C., or about 45° C.For example, a pharmaceutical formulation may be deemed stable if afterthree months of storage at −80° C., −30° C., or −20° C. less than about30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%or 0.1% of the antibody is in a more acidic form. A pharmaceuticalformulation may also be deemed stable if after six months of storage at5° C., less than about 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%,22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a more acidicform. A pharmaceutical formulation may also be deemed stable if aftersix months of storage at 25° C., less than about 43%, 42%, 41%, 40%,39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%,25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody isin a more acidic form. A pharmaceutical formulation may also be deemedstable if after 28 days of storage at 45° C., less than about 49%, 48%,47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%,33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%,19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.5% or 0.1% of the antibody can be detected in a more acidicform.

Other methods may be used to assess the stability of the formulations ofthe present invention such as, e.g., differential scanning calorimetry(DSC) to determine thermal stability, controlled agitation to determinemechanical stability, and absorbance at about 350 nm or about 405 nm todetermine solution turbidities. For example, a formulation of thepresent invention may be considered stable if, after 6 or more months ofstorage at about 5° C. to about 25° C., the change in OD₄₀₅ of theformulation is less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, orless) from the OD₄₀₅ of the formulation at time zero.

Measuring the biological activity or binding affinity of the antibody toits target may also be used to assess stability. For example, aformulation of the present invention may be regarded as stable if, afterstorage at e.g., 5° C., 25° C., 45° C., etc. for a defined amount oftime (e.g., 1 to 12 months), the anti-PCSK9 antibody contained withinthe formulation binds to PCSK9 with an affinity that is at least 90%,95%, or more of the binding affinity of the antibody prior to saidstorage. Binding affinity may be determined by e.g., ELISA or plasmonresonance. Biological activity may be determined by a PCSK9 activityassay, such as e.g., contacting a cell that expresses PCSK9 with theformulation comprising the anti PCSK9 antibody. The binding of theantibody to such a cell may be measured directly, such as e.g., via FACSanalysis. Alternatively, the downstream activity of the PCSK9 system maybe measured in the presence of the antibody, and compared to theactivity of the PCSK9 system in the absence of antibody. In someembodiments, the PCSK9 may be endogenous to the cell. In otherembodiments, the PCSK9 may be ectopically expressed in the cell.

Additional methods for assessing the stability of an antibody informulation are demonstrated in the Examples presented below.

The liquid pharmaceutical formulations of the present invention may, incertain embodiments, exhibit low to moderate levels of viscosity.“Viscosity” as used herein may be “kinematic viscosity” or “absoluteviscosity”. “Kinematic viscosity” is a measure of the resistive flow ofa fluid under the influence of gravity. When two fluids of equal volumeare placed in identical capillary viscometers and allowed to flow bygravity, a viscous fluid takes longer than a less viscous fluid to flowthrough the capillary. For example, if one fluid takes 200 seconds tocomplete its flow and another fluid takes 400 seconds, the second fluidis twice as viscous as the first on a kinematic viscosity scale.“Absolute viscosity”, sometimes called dynamic or simple viscosity, isthe product of kinematic viscosity and fluid density (AbsoluteViscosity=Kinematic Viscosity×Density). The dimension of kinematicviscosity is L²/T where L is a length and T is a time. Commonly,kinematic viscosity is expressed in centistokes (cSt). The SI unit ofkinematic viscosity is mm²/s, which is 1 cSt. Absolute viscosity isexpressed in units of centipoise (cP). The SI unit of absolute viscosityis the milliPascal-second (mPa-s), where 1 cP=1 mPa-s.

As used herein, a low level of viscosity, in reference to a fluidformulation of the present invention, will exhibit an absolute viscosityof less than about 15 cPoise (cP). For example, a fluid formulation ofthe invention will be deemed to have “low viscosity”, if, when measuredusing standard viscosity measurement techniques, the formulationexhibits an absolute viscosity of about 15 cP, about 14 cP, about 13 cP,about 12 cP, about 11 cP, about 10 cP, about 9 cP, about 8 cP, or less.As used herein, a moderate level of viscosity, in reference to a fluidformulation of the present invention, will exhibit an absolute viscosityof between about 35 cP and about 15 cP. For example, a fluid formulationof the invention will be deemed to have “moderate viscosity”, if whenmeasured using standard viscosity measurement techniques, theformulation exhibits an absolute viscosity of about 34 cP, about 33 cP,about 32 cP, about 31 cP, about 30 cP, about 29 cP, about 28 cP, about27 cP, about 26 cP, about 25 cP, about 24 cP, about 23 cP, about 22 cP,about 21 cP, about 20 cP, about 19 cP, 18 cP, about 17 cP, about 16 cP,or about 15.1 cP.

As illustrated in the examples below, the present inventors have madethe surprising discovery that low to moderate viscosity liquidformulations comprising high concentrations of an anti-human PCSK9antibody (e.g., from about 100 mg/ml up to at least 200 mg/mL) can beobtained by formulating the antibody with arginine from about 25 mM toabout 100 mM. In addition, it was further discovered that the viscosityof the formulation could be decreased to an even greater extent byadjusting the sucrose content to less than about 10%.

Containers and Methods of Administration

The pharmaceutical formulations of the present invention may becontained within any container suitable for storage of medicines andother therapeutic compositions. For example, the pharmaceuticalformulations may be contained within a sealed and sterilized plastic orglass container having a defined volume such as a vial, ampule, syringe,cartridge, or bottle. Different types of vials can be used to containthe formulations of the present invention including, e.g., clear andopaque (e.g., amber) glass or plastic vials. Likewise, any type ofsyringe can be used to contain or administer the pharmaceuticalformulations of the present invention.

The pharmaceutical formulations of the present invention may becontained within “normal tungsten” syringes or “low tungsten” syringes.As will be appreciated by persons of ordinary skill in the art, theprocess of making glass syringes generally involves the use of a hottungsten rod which functions to pierce the glass thereby creating a holefrom which liquids can be drawn and expelled from the syringe. Thisprocess results in the deposition of trace amounts of tungsten on theinterior surface of the syringe. Subsequent washing and other processingsteps can be used to reduce the amount of tungsten in the syringe. Asused herein, the term “normal tungsten” means that the syringe containsgreater than or equal to 500 parts per billion (ppb) of tungsten. Theterm “low tungsten” means that the syringe contains less than 500 ppb oftungsten. For example, a low tungsten syringe, according to the presentinvention, can contain less than about 490, 480, 470, 460, 450, 440,430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50,40, 30, 20, 10 or fewer ppb of tungsten.

The rubber plungers used in syringes, and the rubber stoppers used toclose the openings of vials, may be coated to prevent contamination ofthe medicinal contents of the syringe or vial, or to preserve theirstability. Thus, pharmaceutical formulations of the present invention,according to certain embodiments, may be contained within a syringe thatcomprises a coated plunger, or within a vial that is sealed with acoated rubber stopper. For example, the plunger or stopper may be coatedwith a fluorocarbon film. Examples of coated stoppers or plungerssuitable for use with vials and syringes containing the pharmaceuticalformulations of the present invention are mentioned in, e.g., U.S. Pat.Nos. 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, thecontents of which are incorporated by reference herein in theirentireties. Particular exemplary coated rubber stoppers and plungersthat can be used in the context of the present invention arecommercially available under the tradename “FluroTec®”, available fromWest Pharmaceutical Services, Inc. (Lionville, Pa.). FluroTec® is anexample of a fluorocarbon coating used to minimize or prevent drugproduct from adhering to the rubber surfaces.

According to certain embodiments of the present invention, thepharmaceutical formulations may be contained within a low tungstensyringe that comprises a fluorocarbon-coated plunger.

The pharmaceutical formulations can be administered to a patient byparenteral routes such as injection (e.g., subcutaneous, intravenous,intramuscular, intraperitoneal, etc.) or percutaneous, mucosal, nasal,pulmonary or oral administration. Numerous reusable pen or autoinjectordelivery devices can be used to subcutaneously deliver thepharmaceutical formulations of the present invention. Examples include,but are not limited to Autopen® (Owen Mumford, Inc., Woodstock, UK),Disetronic Pen (Disetronic Medical Systems, Bergdorf, Switzerland),Humalog® Mix75/25™ pen, Humalog® pen, Humulin® 70/30 pen (Eli Lilly andCo., Indianapolis, Ind.), NovoPen® I, II and III (Novo Nordisk,Copenhagen, Denmark), NovoPen® Junior (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OptiPen®,OptiPen Pro®, OptiPen Starlet™, and OptiClik® (Sanofi-Aventis,Frankfurt, Germany). Examples of disposable pen or autoinjector deliverydevices having applications in subcutaneous delivery of a pharmaceuticalcomposition of the present invention include, but are not limited to theSoloSTAR® pen (Sanofi-Aventis), the FlexPen® (Novo Nordisk), and theKwikPen™ (Eli Lilly), the SureClick™ Autoinjector (Amgen, Thousand Oaks,Calif.), the Penlet® (Haselmeier, Stuttgart, Germany), the EpiPen® (Dey,L.P.), and the Humira® Pen (Abbott Labs, Abbott Park, Ill.).

The use of a microinfusor to deliver the pharmaceutical formulations ofthe present invention is also contemplated herein. As used herein, theterm “microinfusor” means a subcutaneous delivery device designed toslowly administer large volumes (e.g., up to about 2.5 mL or more) of atherapeutic formulation over a prolonged period of time (e.g., about 10,15, 20, 25, 30 or more minutes). See, e.g., U.S. Pat. No. 6,629,949;U.S. Pat. No. 6,659,982; and Meehan et al., J. Controlled Release46:107-116 (1996). Microinfusors are particularly useful for thedelivery of large doses of therapeutic proteins contained within highconcentration (e.g., about 100, 125, 150, 175, 200 or more mg/mL) orviscous solutions.

In one embodiment, the liquid pharmaceutical formulation containingabout 150 mg/mL±22.5 mg/mL anti-PCSK9 antibody is administeredsubcutaneously in a volume of approximately 1 mL±0.15 ml in a prefilledsyringe. In one embodiment, the syringe is a 1 mL long glass syringefilled with a 27-gauge thin wall needle, a fluorocarbon coated rubberplunger and a rubber needle shield. In one embodiment, the syringe is anOMPI 1 mL long glass syringe fitted with a 27-gauge needle, a FM 27rubber needle shield, and a FluroTec® coated 4023/50 rubber plunger.

In one embodiment, the liquid pharmaceutical formulation containingabout 150 mg/mL±22.5 mg/mL anti-PCSK9 antibody is administeredsubcutaneously in a volume of approximately 1 mL±0.15 ml in a prefilledsyringe. In one embodiment, the syringe is a 1 mL long glass syringefilled with a 27-gauge thin wall needle, a fluorocarbon coated rubberplunger and a rubber needle shield. In one embodiment, the syringe is anOMPI 1 mL long glass syringe fitted with a 27-gauge needle, a FM27rubber needle shield, and a FluroTec® coated 4023/50 rubber plunger.

Therapeutic Uses of the Pharmaceutical Formulations

The pharmaceutical formulations of the present invention are useful,inter alia, for the treatment, prevention or amelioration of any diseaseor disorder associated with PCSK9 activity, including diseases ordisorders mediated by PCSK9. Exemplary, non-limiting diseases anddisorders that can be treated or prevented by the administration of thepharmaceutical formulations of the present invention include variousdyslipidemias such as, e.g., hypercholesterolemia, familialhypercholesterolemia, hyperlipidemia, familial hyperlipidemia,dysbetalipoproteinemia, familial dysbetalipoproteinemia,hypertriglyceridemia, and familial hypertriglyceridemia.

EXAMPLES

The following examples are presented so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by mole, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric pressure.

Initial formulation development activities involved screening organiccosolvents, thermal stabilizers, and buffers in liquid and lyophilizedformulations of mAb-316P (anti-PCSK9 antibodies of the invention) toidentify excipients that are compatible with the protein and enhance itsstability, while maintaining near physiologic osmolality and lowviscosity for intravenous and subcutaneous injection. Buffer conditionswere also examined to determine the optimal pH for maximum proteinstability.

Example 1 Development of Anti-PCSK1 MAB-316P Formulation

Various buffers, organic cosolvents, and thermal stabilizers werescreened to identify excipients that enhance the stability of the PCSK9antibody. Buffer conditions were also examined to determine the optimalpH for maximum antibody stability. Results generated from these studieswere used to develop a stable liquid formulation, as well as a stablelyophilized formulation suitable for clinical use, for eitherintravenous (IV) or subcutaneous administration (SC). For thelyophilized drug product, a single, dual use formulation was developedwhich can be reconstituted with sterile water for injection (WFI) to aconcentration of either 50 mg/mL for IV or 100 mg/mL for SCadministration. Once reconstituted to 50 mg/mL, the drug product can befurther diluted into an IV bag containing 0.9% sodium chloride for IVdelivery. For the liquid formulation, mAb-316P was formulated at 175±27mg/ml and 150±23 mg/ml. In one embodiment, the 175±27 mg/mL mAb-316P isformulated in 10±1.5 mM histidine (pH 6.0±0.3), 0.01%±0.0015%polysorbate 20, 5%±0.75% sucrose. In one embodiment, the 150±23 mg/mLmAb-316P is formulated in 10±1.5 mM histidine (pH 6.0±0.3), 0.2%±0.03%or 0.01%±0.0015% polysorbate 20, 10%±1.5% sucrose.

Example 2 Anti-PCSK1 MAB-316P Buffer and pH

The effect of pH and buffer type on the stability of the PCSK9antibodies was examined in liquid formulations. 2 mg/mL anti-PCSK9mAb-316P was incubated at 45° C. in 10 mM each of either acetate (pH5.0-5.5), citrate (pH 5.5-6.0), succinate (pH 6.0), histidine (pH 6.0),phosphate (pH 6.0-7.5), or Tris (pH 8.0) buffer to assess the effect ofbuffer and pH on the thermal stability of the protein (Table 1). Forthis experiment, the liquid formulations were each kept as 0.35 mL in a2 mL capacity Type 1 borosilicate glass vial with a FluroTec® coated4432/50 butyl rubber stopper. The total amount of mAb-316P recovered wasdetermined using reverse-phase chromatography. The percentage of thenative versus aggregated form of mAb-316P was determined usingsize-exclusion chromatography. The percentage of acidic and basicspecies of the mAb-316P was determined using cation exchangechromatography. Maximum protein stability was observed, as determined byboth size exclusion chromatography (SE) and cationic exchangechromatography (CEX), when anti-PCSK9 mAb-316P was formulated in 10 mMhistidine buffer at pH 6.0.

The optimal pH for mAb-316P was then determined by incubating 10 mg/mLof mAb-316P at 45° C. in histidine buffer between pH 5.5 and pH 6.5.Maximum protein stability was observed, as determined by SE and CEX,when mAb-316P was formulated in histidine buffered at pH 6.0 (Table 2).These analyses also revealed that the main protein degradation pathwayswere the formation of aggregates, cleavage products, and chargevariants. Based on these results, 10 mM histidine buffer at pH 6.0 waschosen for development of a liquid and lyophilized mAb-316P formulation.

Results from formulation development studies indicate that under basicconditions (pH 6.5), anti-PCSK9 mAb-316P in solution may undergodeamidation reactions. Conversely, at pH 5.5, an increased rate offormation of molecular weight variants of mAb-316P was observed. Basedon these data, the buffer pH used for the formulation of the mAb-316P ismaintained between pH 5.7 and pH 6.3. The accelerated stability ofmAb-316P is similar over this pH range.

Example 3 Selection of Protectants Against Agitation Stress

Various cosolvents were individually tested for their ability tominimize the formation of particulates in mixtures containing mAb-316Pdue to agitation stress. Turbidity analysis of agitated drug substancedemonstrated an increase in the optical density (OD) at 405 nm when asolution containing mAb-316P (0.35 mL of 25 mg/mL mAb-316P, 10 mMhistidine, pH 6.0±0.2 in a 2 mL capacity Type 1 borosilicate glass vialwith a FluroTec® coated 4432/50 butyl rubber stopper) was vortexed for120 minutes (Table 3). Formulation with any of the evaluated cosolventsappears to have prevented the agitation-induced increase in turbidity.However, 20% PEG 300, 10% PEG 300, and 20% propylene glycolsignificantly decreased the thermal stability of mAb-316P as determinedby SE (Table 4; same mAb-316P concentration, buffer, and containerconditions as in the vortex study above). Formulations with polysorbate20, polysorbate 80, Pluronic® F-68, and PEG 3350 had no significanteffect on the thermal stability of mAb-316P as determined by SE and CEX,making these cosolvents suitable for formulating anti-PCSK9 mAb-316P.Polysorbate 20 was chosen as the organic cosolvent for development ofboth a lyophilized and liquid formulation of mAb-316P because itdemonstrated good stability attributes in both the agitation and thermalstudies of mAb-316P.

Example 4 Selection of Protectants Against Thermal Stress

Various excipients, which were selected from a varied list containingsugars, amino acids, and inorganic salts, were individually tested tooptimally increase the thermal stability of mAb-316P. A summary of somethe thermal stabilizers that were examined is presented in Table 5. Forthese experiments, the “thermal stabilizer” excipients were included ina solution of 20 mg/mL mAb-316P in 10 mM histidine (0.35 mL in 2 mLcapacity Type 1 borosilicate glass vial with a FluroTec® coated 4432/50butyl rubber stopper). Formulations containing sucrose, sorbitol,mannitol, and trehalose had the least amount of mAb-316P degradation asdetermined by SE analysis. However, those formulations containingsorbitol showed a surprising increase in turbidity compared to thoseformulations containing sucrose, trehalose, and mannitol (Table 5).While sucrose, trehalose, and mannitol were observed to have no effecton the formation of charge variants of the anti-PCSK9 mAb-316P, mannitolwas observed to destabilize the protein during multiple freeze-thawcycles. Thus, mAb-316P has similar stability when formulated withsucrose or trehalose.

Example 5 Lyophilized Formulation

A lyophilized formulation was developed to increase the stability ofanti-PCSK9 mAb-316P, particularly with respect to charge variants, andto increase the maximum deliverable concentration of mAb-316P. Variouslyoprotectants were combined with 0.7 mL of 50 mg/mL mAb-316P, 10 mMhistidine, in a 2 mL capacity Type 1 borosilicate glass vial with aFluroTec® coated 4432/50 butyl rubber stopper, lyophilized, and examinedfor their ability to stabilize lyophilized mAb-316P when incubated at50° C. Prior to analysis, the lyophilized cake was reconstituted to 100mg/mL mAb-316P. The two lyophilized formulations with the greateststability as determined by SE and CEX contained: 1) 6% sucrose or 2) 2%sucrose plus 2% arginine. 6% sucrose was chosen for the mAb-316P drugproduct formulation. Thus, the anti-PCSK9 mAb-316P lyophilized drugproduct was produced by lyophilization in an optimized, aqueous bufferedformulation containing 10 mM histidine, pH 6.0±0.1, 0.1% (w/v)polysorbate 20, 6% (w/v) sucrose, and 50 mg/mL anti-PCSK9 mAb-316P. Thestorage and stress stability of this lyophilized formulation ispresented in Table 7.

The anti-pPCSK9 mAb-316P lyophilization cycle was developed based on themeasured Tg′ (frozen glass transition temperature) of the formulation,which was measured using subambient modulated differential scanningcalorimetry (mDSC). The product temperature must not go above the Tg′during primary drying, which was determined to be −27.9° C.

Lyophilized mAb-316P was produced by filling 5.3 mL of the 50 mg/mLmAb-316P, 10 mM histidine (pH 6.0), 0.1% polysorbate 20, 6% sucroseformulation into 20 mL Type 1 glass vials and lyophilizing according tothe following steps:

1. Shelf temperature required during loading: 5-25° C.

2. Initial Hold at: 5° C. for 60 minutes

3. Ramp rate (time) for freezing: 0.5° C./min (100 minutes)

4. Hold at: −45° C. for 120 minutes

5. Vacuum Set Point: 100 mTorr

6. Ramp rate (time) for heating to primary drying: 0.5° C./min (40minutes)

7. Shelf Temperature of Primary Drying: −25° C.

8. Length of Primary Drying: 78 hours

9. Ramp rate (time) for heating to secondary drying: 0.2° C./min (300minutes)

10. Shelf temperature of Secondary Drying: 35° C.

11. Length of Secondary Drying: 6 hours

12. Ramp rate (time) for cooling: 0.5° C./min (20 minutes)

13. Hold at: 25° C. for 60 minutes*

14. Backfill with nitrogen gas

15. Stoppering under vacuum: 80% of Atmospheric pressure (608,000 mTorr)

When extensive storage is needed after secondary drying and prior to thestoppering step, the shelf temperature of the lyophilizer is brought to2-8° C. Lyophilized drug product that was produced using the finalcycles described above had good cake appearance, low moisture content(0.3%), reconstitution time less than 4 minutes, and no turbidity of thereconstituted solution.

The appearance of the lyophilized cake was unaffected when the mAb-316Pdrug product (mAb-316P DP) was incubated for 2 months at 50° C. orstored for 3 months at 5° C. There was no affect on pH, appearance, orturbidity of the reconstituted mAb-316P drug product, and no significantdifference in the amount of mAb-316P recovered. After 2 months ofincubation at 50° C., the lyophilized mAb-316P drug product was 1.1%more degraded as determined by SE-HPLC and 8.3% more degraded asdetermined by CEX-HPLC. No significant degradation was observed when thelyophilized mAb-316P drug product was stored for 3 months at 5° C. Nosignificant loss of potency, as determined using an anti-PCSK9 bioassay,was observed for any of the stressed samples.

Example 6 Liquid and Reconstituted MAB-316P

There are two methods to reconstitute lyophilized mAb-316P drug productdepending on the route of administration. For IV administration,mAb-316P drug product is reconstituted with 5.0 mL of sterile WFIresulting in 5.3 mL of solution containing 50 mg/mL mAb-316P, 10 mMhistidine, pH 6.0, 0.1% (w/v) polysorbate 20, and 6% (w/v) sucrose. ForSC administration, mAb-316P drug product is reconstituted with 2.3 mL ofsterile WFI resulting in 2.7 mL solution containing 100 mg/mL REGN727,20 mM histidine, pH 6.0, 0.2% (w/v) polysorbate 20, and 12% (w/v)sucrose. The volume available for withdrawal is 4.8 mL for IV and 2.0 mLfor SC injection; an overage of 0.7 mL of reconstituted solution iscontained in the SC vial.

In the alternative, liquid mAb-316P is formulated as a liquidformulation, without the intervening step of lyophilization. The liquidmAb-316P formulations are at either 150 mg/mL (±15%) or 175 mg/mL (±15%)anti-PCSK9 mAb-316P, in 10±1.5 mM histidine (pH 6.0±0.3), polysorbate 20at 0.01%±0.0015% or 0.2%±0.03%, sucrose at 5%±0.75% or 10%±1.5%, and, inthe case of the 175 mg/mL formulation, arginine at 50±7.5 mM.

Anti-PCSK9 mAb-316P was found to be stable when sterile filtered. AMillipore Millipak® filtration unit was used in the manufacture of theclinical supplies, while a filter of identical composition was used inresearch studies (Millipore Millex® Durapore®). Compared to storage inglass vials, the stability of mAb-316P formulated drug substance(mAb-316P FDS) was not significantly affected when stored in either apolypropylene tube, a polystyrene tube, a polycarbonate tube, or in aglass vial containing a stainless steel ball bearing (Table 8).

Example 7 Liquid Formulation in Prefilled Syringes

Formulation development studies were conducted with the goal ofdeveloping a high concentration, liquid formulation of mAb-316P thatcould be used in pre-filled syringes (PFS) for SC delivery. Results fromthe development phase of the lyophilized REGN727 formulationdemonstrated that the optimal buffer, pH, organic cosolvent, and thermalstabilizer were histidine, pH 6.0, polysorbate 20, and sucrose,respectively (supra). These same excipients were used to develop boththe 150 mg/mL and 175 mg/mL mAb-316P drug product formulations. Argininewas added to the 175 mg/mL version of the mAb-316P drug product toreduce the viscosity of the formulation. The stress stability of 150 and175 mg/mL mAb-316P drug product was examined in 1 mL long, glass NuovaOmpi Pre-filled Syringes (PFS) and compared to the stability of 150 and175 mg/mL drug product in control, glass vials. No significantdifference in the amount of physical or chemical degradation wasobserved after incubation of 150 or 175 mg/mL mAb-316P drug product at45° C. between the OMPI PFS and the glass control vial. These dataindicate that the 150 and 175 mg/mL anti-PCSK9 mAb-316P drug productformulations are sufficiently stable for use in PFS.

Example 8 Stability of MAB-316P Formulated Drug Substance

Stability studies were performed to determine both the storage andstress stability of 150 and 175 mg/mL mAb-316P formulations. Turbidityand RP-HPLC assays were used to assess the physical stability ofmAb-316P. Physical stability is defined as the recovery of soluble formsof the anti-PCSK9 mAb-316P in solution. Loss of protein could be due toeither protein precipitation or surface adsorption. The presence ofparticulates in solution can be detected by visual inspection or byoptical density (OD) measurements at 405 nm (turbidity measurements). Inthis latter assay, an increase in OD indicates an increase in turbiditydue to the formation of particulates. The presence of particulates asdetermined by OD measurements indicates that the sample has failed tomaintain stability. Recovery of mAb-316P is measured by RP-HPLC. In theRP-HPLC assay, the ant-PCSK9 mAb-316P antibody is eluted from thereverse phase column as a single peak. The concentration of each testsample is determined from the area of the eluted mAb-316P antibody peakcompared to a calibration curve generated using mAb-316P standards ofdefined protein loads.

Chemical stability refers to the integrity of the chemical structure ofthe anti-PCSK9 antibody (mAb-316P) in a sample. Most chemicalinstability can be attributed to the formation of covalently modifiedforms of the protein, (e.g. covalent aggregates, cleavage products, orcharge variants) and non-covalently modified forms of the protein (e.g.non-covalent aggregates). Thus far, the only degradation products ofmAb-316P that have been detected are species that differ in eithermolecular weight or charge. The higher and lower molecular weightdegradation products can be separated from native mAb-316P by SE-HPLC.The percentage of native mAb-316P in the size exclusion chromatographicmethod is determined by the ratio of the area of the native peak to thetotal area of all mAb-316P antibody peaks.

Charge variant forms of mAb-316P are resolved from native mAb-316P usingcation exchange chromatography. Peaks that elute from the CEX-HPLCcolumn with retention times earlier than that of the main peak arelabeled “Acidic Peaks”, while those that elute from the CEX-HPLC columnwith retention times later than that of the main peak are labeled “BasicPeaks”. The percentage of degraded mAb-316P in the cation exchangechromatographic method is determined by the change in the relativepercentage of the main, acidic, and basic peak areas compared to thetotal area of all mAb-316P peaks.

Evaluation of mAb-316P under accelerated conditions was performed bysubjecting the antibody to a variety of stress tests. These testsrepresent the extreme handling conditions that the formulated drugsubstance may be subjected to during the manufacture of drug product.mAb-316P formulated drug substance was filled in 5 mL polycarbonatevials for the agitation, cycles of freeze/thaw, and frozen storageconditions. mAb-316P formulated drug substance was filled in glass vialsto examine stress stability at high temperatures.

Example 9 Storage Stability Studies of Formulated Drug Substance (FDS)

150 mg/mL mAb-316P formulated drug substance (FDS; 0.5 mL in 5 mLpolycarbonate vial; 150 mg/mL mAb-316P antibody, 10 mM histidine (pH6.0), 0.2% polysorbate 20, and 10% sucrose) was found to be physicallyand chemically stable when stored at −20° C. for 12 months. Nosignificant loss of mAb-316P was observed and no significant chemicaldegradation was detected by size exclusion or ion exchangechromatography. Greater than 97% of the recovered mAb-316P was of the“native” structure as determined by size exclusion, and greater than 56%of the recovered mAb-316P was of the “main charge variant” as determinedby cation exchange. The results are summarized in Table 9.

175 mg/mL mAb-316P formulated drug substance (FDS; 0.75 mL in 5 mLpolycarbonate vial; 175 mg/mL mAb-316P antibody, 10 mM histidine (pHs6.0), 0.01% polysorbate 20, 5% sucrose, and 50 mM arginine) was found tobe physically and chemically stable when stored at ≦−20° C. for 3months. No significant loss of mAb-316P was observed and no significantchemical degradation was detected by size exclusion or ion exchangechromatography. Greater than 96% of the recovered mAb-316P was of the“native” structure as determined by size exclusion, and greater than 56%of the recovered mAb-316P was of the “main charge variant” as determinedby cation exchange. The results are summarized in Table 10.

Example 10 Stress Stability Studies of Formulated Drug Substance

Stress stability studies were performed on the 150 mg/mL mAb-316Pformulated drug substance (FDS) (0.35 mL-0.5 mL of 150 mg/mL mAb-316P,10 mM histidine (pH 6.0), 0.2% polysorbate 20, 10% sucrose) and the 175mg/mL mAb-316P formulated drug substance (0.5 mL-1.7 mL of 175 mg/mLmAb-316P, 10 mM histidine (pH 6.0), 0.01% polysorbate 20, 5% sucrose, 50mM arginine). High temperature studies were conducted in a 2 mL capacityType 1 borosilicate glass, FluroTec® coated 4432/50 butyl rubberstopper; the remaining studies were performed in a 5 mL polycarbonatevial. The 150 mg/mL and the 175 mg/mL anti-PCSK9 mAb-316P formulateddrug substance were found to be physically and chemically stable whenagitated (vortexed) for two hours. The solution remained visibly clear,no loss of protein occurred, and no molecular weight species or chargevariants were formed (Tables 11 & 12). MAb-316P was also observed to beboth physically and chemically stable when subjected to eight cycles offreezing to −80° C. and thawing to room temperature. Following the eightfreeze/thaw cycles, the protein solution remained visibly clear and noloss of protein was observed. No molecular weight (either solubleaggregates or cleavage products) or charge variant forms were detectedby either SE or CEX assays, respectively.

Although the 150 and the 175 mg/mL anti-PCSK9 mAb-316P formulated drugsubstance were physically stable when incubated at 37° C. or 45° C. for28 days, some chemical degradation was nonetheless observed (Tables 11 &12). These stress tests indicated that the main degradation pathwayswere the formation of aggregates, cleavage products, and chargevariants. As expected, the rate of degradation of anti-PCSK9 mAb-316Pantibody was slower at 37° C. than at 45° C. There was no significantchange in the physical or chemical stability of 150 or 175 mg/mLmAb-316P formulated drug substance when incubated at 25° C. for 28 days.

Example 11 Storage Stability of Drug Product (DP)

The 150 mg/mL mAb-316P drug product consists of 10 mM histidine, pH 6.0,0.01% polysorbate 20, 10% sucrose, and 150 mg/mL anti-PCSK9 mAb-316Pantibody. The 175 mg/mL mAb-316P drug product consists of 10 mMhistidine, pH 6.0, 0.01% polysorbate 20, 5% sucrose, 50 mM arginine, and175 mg/mL anti-PCSK9 mAb-316P antibody. There was no change in thephysical and chemical stability of either the 150 mg/mL or the 175 mg/mLmAb-316P drug product (DP) when stored at 5° C. for 6 months in thepre-filled syringe (PFS; OMPI 1 mL long lass syringe with a 27 gaugethin wall needle and FM 27 rubber needle shield closed with a FluroTec®coated 4023/50 rubber plunger) (Table 13 and Table 14). The solutionsremained visibly clear, no loss of protein was observed, and no changein pH occurred after these stresses. In addition, there was nosignificant change in molecular weight species or charge variants weredetected by SE and CEX, respectively.

Example 12 Stress Stability of Drug Product (DP)

The stress stabilities of 150 mg/mL mAb-316P drug product and 175 mg/mlmAb-316P drug product were examined by incubating the pre-filledsyringes at 25° C. and 45° C. Each respective drug product wasphysically stable when incubated at 45° C. for 28 days or incubated at25° C. for 6 months (Tables 13 & 14). The solution remained visiblyclear, no loss of protein was observed, and no change in pH occurredafter these stresses. However, aggregates and charge variants weredetected when the protein was incubated at 45° C. and 25° C. This stresstest indicates these are the main degradation pathways for drug product.Of the 150 mg/mL drug product, the mAb-316P aggregate increased 1.9% andacidic species increased 19.1% after incubation at 45° C. for 28 days. Areduced level of chemical degradation was detected when the protein wasincubated at 25° C. There was a 0.8% increase in the relative amount ofaggregate and an 10.3% increase in acidic species after 6 months ofincubation at 25° C. Of the 175 mg/mL drug product, the mAb-316Paggregate increased 1.8% and acidic species increased 17.0% afterincubation at 45° C. for 28 days. A reduced level of chemicaldegradation was detected when the protein was incubated at 25° C. Therewas 0.7% increase in aggregate and a 9.4% increase in acidic speciesafter 6 months of incubation at 25° C. For both the 150 and 175 mg/mLdrug products, there was no significant change in the stability ofmAb-316P after 1 month of incubation at 25° C.

Example 13 Fill Volumes

The injectable volume from a pre-filled syringe (PFS) containing 150mg/mL REGN727 drug product is 1.0 mL. The injectable volume from a PFScontaining 175 mg/mL REGN727 drug product is 1.14 mL. No overage isincluded in either PFS because the dead volume in the syringe isnegligible (0.005 to 0.01 mL).

Example 14 Stability of MAB-316P in Storage Materials

Anti-PCSK9 mAb-316P was found to be stable when sterile filtered. AMillipore Millipak® filtration unit was used for research studies and inthe manufacture of the clinical supplies. Compared to storage in glassvials, the stability of 150 and 175 mg/mL mAb-316P formulated drugsubstance was not significantly affected when stored in a polypropylenetube, a polystyrene tube, a polycarbonate tube, or in a glass vialcontaining a stainless steel gasket (Table 15 and Table 16). Althoughdegradation was observed when the formulated drug substance wasincubated at 40° C. for 14 days, no significant difference in the amountof mAb-316P degradation was observed between the control, glass vial andexposure to the plastic containers and stainless steel.

Example 15 Characterization of Anti-PCSK9 Antibody MAB-316P

At least two lots of mAb-316P (Lot 1 and Lot 2) were analyzed by sizeexclusion chromatography and multi-angle laser light scattering(SEC-MALLS), an analytical method that gives an estimate of the molarmass of a protein or glycoprotein. Lots 1 and 2 had respective molarmasses of 154.5 and 154.6 kDa. Other lots had molar masses ranging from154.4 to 154.8 kDa (average of about 155 kDa) for the main species peakeluted from the SE matrix. This main peak represented about 96.7-99.2%of the total protein peak area and corresponds to intact mAb-316Pmonomer (i.e., “native” as used herein).

mAb-316P was analyzed by capillary isoelectric focusing (cIEF) todetermine the isoelectric points for the major constituent isoforms. Thepl and average peak area (% total peak area) of mAb-316P samplesdetermined by clEF are summarized in Table 17. Each lot exhibited a mainspecies (peak 5) with a calculated pl of approximately 8.5, which waspresent at 66.4% and 68.0% for lots 1 and 2, respectively. The dominantspecies (peak 5) most likely represents intact fully glycosylatedantibody lacking the C-terminal lysine (i.e., “main charge form” as usedherein).

Mass spectrometric (MS) analysis of the reduced mAb-316P tryptic mapsfrom Lot 1 and Lot 2 resulted in the confirmation of a singleglycosylation site, Asn298, within the Fc domain in both lots. The majorcovalently linked glycan forms this glycosylation site are summarized inTable 18. Overall, both lots were determined to possess complexbi-anntennary glycans, with majority of them fucosylated at Asn298.However, the relative amount of fucosylated agalactosyl (G0) containingsugar chain species in Lot 2 was slightly higher relative to the amountof this glycoform in Lot 1. Conversely, the relative amounts offucosylated digalactosyl (G2) and fucosylated monogalactosyl (G1)containing sugar chain forms in Lot 2 were reduced compared to therelative amounts of these sugar chain structures in Lot 1. Analysis ofthe LC/MS results of the two drug substance samples (peak 16) alsoidentified 2.9% and 8.4% of heavy chain peptide lacking glycan occupancyat Asn298 on Lot 1 and Lot 2, respectively.

Glycan profiles generated by HPLC after release of oligosaccharides fromeach of the two mAb-316P lots were analyzed. In each chromatogram, thederivatized oligosaccharides were separated into two main groups:non-fucosylated bi-antennary species and fucosylated bi-antennaryspecies. Within each group (fucosylated vs. non-fucosylated), theoligosaccharides were further separated into digalactosyl (G2),monogalactosyl (G1), and agalactosyl (G0) forms. Oligosaccharidestructure assignments were obtained via MALDI-TOF mass spectrometry.Integration of each peak in the two chromatograms revealed that thefucosylation level of the two mAb-316P lots was generally high, with80.0% and 86.9% fucosylation observed in Lots 1 and 2, respectively.

Although the total percent fucosylation of the two lots was similar,there were quantitative differences in the relative abundance of each ofthe glycan forms present in the two lots (Table 19). For Lot 1, peakarea percentages of 34.4%, 39.6%, and 11.7%, were determined for the G0,G1, and G2 fucosylated glycan structures, respectively. In contrast,peak area percentages for Lot 2 were 45.8%, 33.3%, and 7.1%, for the G0,G1, and G2 fucosylated glycoform structures, respectively, indicatingdifferences in the extent of galactosylation between the two lots. Ahigh mannose glycan (man5 glycan) peak (peak 2) was detected at arelative abundance of 1.8-2.9% relative to the total amount of sugarchain observed in both lots (Table 19). A total of nine unidentifiedpeaks with peak area percentages ranging from 0.5% to 1.5% were alsodetected in both lots examined by this method and represent ≦3% of thetotal glycan peak areas detected in each lot. Analysis of an equimolarco-mixture of the two lots yielded no new peaks and the peak areapercentages of all peaks in the co-mixture correlated well with expectedvalues based on individual analysis of each lot (Table 19).

TABLE 1 Effect of buffer and pH on mAb-316P stability at 45° C. for 28days Total pH/Buffer Turbidity¹ (mg/mL) % Native % Aggr. % Main % Acidic% Basic no incubation² 0.00 1.8 98.1 0.3 57.0 33.0 10.0 pH 8.0, Tris0.00 1.7 88.9 0.8 15.2 82.4 2.4 pH 8.0, 0.01 2.0 89.4 1.3 NA NA NAPhosphate pH 7.5, 0.01 1.9 91.7 0.9 NA NA NA Phosphate pH 7.0, 0.01 2.192.4 0.7 16.7 78.8 4.5 Phosphate pH 6.5, 0.00 2.0 93.2 0.6 25.0 68.3 6.6Phosphate pH 6.0, 0.00 1.8 93.9 0.3 29.8 62.3 8.0 Phosphate pH 6.0, 0.001.9 94.5 0.0 36.9 54.0 9.1 Histidine pH 6.0, 0.00 1.9 93.1 0.4 31.9 59.88.3 Succinate pH 6.0, Citrate 0.00 1.9 95.1 0.4 32.1 58.1 9.9 pH 5.5,Citrate 0.00 2.1 94.6 0.4 28.0 62.0 9.9 pH 5.5, Acetate 0.00 2.0 92.40.2 34.5 56.9 8.5 pH 5.0, Acetate 0.00 1.8 93.0 0.2 31.1 57.5 11.4¹Turbidity = change in OD at 405 nm relative to starting material.²Average values of the non-incubated material for all 12 formulations

TABLE 2 Effect of pH on 10 mg/mL mAb-316P, 10 mM Histidine at 45° C. for28 Days Total % % % % % pH/Buffer Turbidity¹ mg/mL Native Aggregate MainAcidic Basic no 0.00 9.5 98.1 0.2 57.8 31.1 11.1 incubation² pH 5.5 0.019.5 94.3 0.5 34.7 52.2 13.1 pH 6.0 0.01 9.7 94.7 0.9 37.5 51.8 10.6 pH6.5 0.01 10.1 93.4 1.8 35.7 55.2 9.1 ¹Turbidity = change in OD at 405 nmrelstive to starting material. ²Average values of the non-incubatedmaterial for all 3 formulations

TABLE 3 Effect of Cosolvents on 25 mg/mL mAb-316P Vortexed for 120Minutes Organic Total Cosolvent Turbidity¹ (mg/mL) % Native % Agg'd %Main % Acidic % Basic No vortexing² 0.00 25.6 97.3 0.5 50.6 39.2 10.3 NoCosolvent 0.10 25.6 97.3 0.5 51.1 39.0 9.9 0.2% 0.01 24.7 97.2 0.5 51.138.9 10.1 Polysorbate 20 0.2% 0.01 24.7 97.3 0.5 50.9 38.9 10.2Polysorbate 80 0.2% Pluronic 0.01 25.2 96.9 0.5 50.5 39.2 10.3 F68 3%PEG 3350 0.01 25.3 97.1 0.5 50.7 39.0 10.2 1.5% PEG 0.01 24.9 97.1 0.550.8 39.1 10.1 3350 20% PEG 300 0.00 26.7 97.0 0.6 48.8 40.5 10.7 10%PEG 300 0.01 25.7 97.2 0.5 49.9 39.8 10.3 20% Propylene 0.01 25.8 96.90.5 51.1 38.6 10.3 Glycol ¹Turbidity = change in OD at 405 nm relstiveto starting material. ²Average values of the non-vortexed material forall 9 formulations

TABLE 4 Effect of Cosolvents on 25 mg/mL mAb-316P Incubated at 45° C.for 28 Days Organic Total % % % % % Cosolvent Turb'ty¹ pH mg/mL NativeAgg'd Main Acidic % Basic no incubation² 0.00 6.1 25.6 97.3 0.5 50.639.2 10.3 No Cosolvent 0.02 6.2 24.9 94.5 0.7 34.8 54.7 10.5 0.2% 0.036.2 24.3 94.6 0.5 35.2 54.3 10.5 Polysorbate 20 0.2% 0.02 6.2 24.3 94.80.6 35.1 54.5 10.4 Polysorbate 80 0.2% Pluronic 0.03 6.1 24.6 94.7 0.633.8 55.6 10.6 F68 3% PEG 3350 0.03 6.2 24.8 95.0 0.7 35.9 53.5 10.61.5% PEG 0.02 6.2 24.4 94.8 0.6 36.0 53.5 10.5 3350 20% PEG 300 0.12 4.825.4 88.8 4.2 NA NA NA 10% PEG 300 0.09 5.4 25.0 93.7 0.9 24.1 67.0 8.920% Propylene 0.03 6.1 24.6 89.9 5.6 34.6 54.2 11.2 Glycol ¹Turbidity =change in OD at 405 nm relstive to starting material. ²Average values ofthe non-vortexed material for all 9 formulations

TABLE 5 Effect of stabilizer on 20 mg/mL mAb-316P, 10 mM histidine at45° C. for 28 days Total % % % % % Excipient Visual Turb'ty¹ pH mg/mLNative Agg'd Main Acidic Basic no incubation² Pass 0.00 6.0 20.2 97.70.5 51.1 39.3 9.6 No Stabilizer Pass 0.02 6.1 19.8 93.6 2.0 33.5 56.510.1 150 mM NaCl Fail 0.03 6.0 20.0 91.0 4.6 35.3 51.0 13.6 20% SucrosePass 0.04 6.0 22.6 94.4 1.0 32.1 57.0 10.9 20% Sorbitol Pass 0.16 5.821.8 94.3 1.0 23.5 67.4 9.1 10% Mannitol Pass 0.02 6.0 21.2 94.8 0.934.4 54.5 11.1 20% Pass 0.05 6.0 22.7 94.7 0.5 33.2 56.6 10.2 Trehalose5% Glycerol Pass 0.10 5.9 20.8 90.2 5.4 NA NA NA 3% Arginine Pass 0.026.1 21.1 92.9 3.0 37.5 49.2 13.3 3% Glycine Pass 0.03 6.1 19.8 93.7 1.731.0 58.0 10.9 ¹Turbidity = change in OD at 405 nm relative to startingmaterial. ²Average values of the non-incubated material for all 9formulations

TABLE 6 Effect of lyoprotectants on lyophilized mAb-316P incubated at50° C. for 28 days Total % % % % % Excipient Visual Turb'ty¹ pH mg/mLNative Agg'd Main Acidic Basic no incubation Pass 0.00 6.0 100 97.1 1.251.0 38.3 10.7 No Lyoprotectant Fail 0.09 6.1 100 70.0 28.5 24.2 35.140.7 2% Sucrose Pass 0.02 6.1 104 90.7 7.4 37.4 39.1 23.6 6% SucrosePass 0.00 6.1 105 96.1 1.8 46.8 39.2 14.1 2% Sucr., 2% Gly Pass 0.02 6.0114 94.5 3.4 40.9 42.4 16.8 2% Sucr., 2% Arg Pass 0.00 5.9 109 95.9 2.047.2 38.4 14.4 ¹Turbidity = change in OD at 405 nm relstive to startingmaterial. ²Lyophilized Drug Product reconstituted to 100 mg/mL REGN727prior to analysis. ³Average values of the starting material of 4formulations.

TABLE 7 Stability of lyophilized Drug Product Reconstituted to 100 mg/mlStorage Temperature No 5° C. 25° C. 40° C. 50° C. storage 4 mo. 3 mo. 3mo. 2 mo. Turbidity¹ 0.00 0.01 0.01 0.02 0.02 pH 6.2 6.3 6.2 6.2 6.2 %Total Recvr'd 100 104 100 98 105 % Native 96.0 96.5 96.2 95.7 94.9 %Aggregate 1.7 1.4 1.7 2.4 2.9 % Main 50.6 51.5 49.2 46.2 43.5 % Acidic38.0 37.9 38.2 39.5 40.1 % Basic 11.4 10.5 12.7 14.3 16.2 Bioassay (%Ref. Std.)² 146 NP NP NP 152 ¹Turbidity = change in OD at 405 nmrelative to starting material. ²Acceptance criteria: 50-200% ofreference standard

TABLE 8 Compatibility of 50 mg/mL mAb-316P for 14 days at 40° C. and 75%humidity No Poly- Poly- Poly- Stainless Storage Storage Glass propylenestyrene carbonate Steel Turbidity¹ 0.00 0.00 0.01 0.00 0.01 0.01 % Total100 98 99 104 103 98 % Native 97.0 96.3 96.3 96.2 96.2 96.1 % Aggregate2.0 1.9 1.9 2.0 2.0 2.0 % Main 50.8 45.7 44.9 45.2 45.6 45.5 % Acidic38.1 42.8 43.6 43.0 42.5 43.2 % Basic 11.2 11.5 11.6 11.9 11.8 11.2¹Turbidity = change in OD at 405 nm relative to starting material.

TABLE 9 Stability of 150 mg/mL anti-PCSK9 mAb-316P for 12 months-80° C.Storage Temperature control −80° C. −30° C. −20° C. Turbidity (OD 405nm) 0.00 0.00 0.01 0.01 % Total mAb-316P 100 104 108 111 Recovered %Native Recovered 97.5 97.3 97.2 97.2 % Aggregate Recovered 1.7 1.8 1.81.9 % Main Recovered 56.2 56.5 56.4 56.3 % Acidic Recovered 26.5 25.725.7 25.4 % Basic Recovered 17.4 17.9 17.9 18.2

TABLE 10 Stability of 175 mg/mL anti-PCSK9 mAb-316P for 3 months-80° C.Storage Temperature control −80° C. −30° C. −20° C. Turbidity (OD 405nm)¹ 0.00 0.01 0.01 0.01 % Total REGN727 Recovered 100 100 104 101(RP-HPLC) % Native REGN727 Recovered 96.2 96.5 96.4 96.3 (SE-HPLC) %REGN727 Aggregate Recovered 2.7 2.5 2.6 2.7 (SE-HPLC) % Main REGN727Recovered 58.5 56.2 56.6 56.7 (CEX-HPLC) % Acidic REGN727 Recovered 29.429.4 29.4 29.4 (CEX-HPLC) % Basic REGN727 Recovered 12.2 14.5 14.0 13.9(CEX-HPLC) ¹Turbidity = change in OD at 405 nm relative to startingmaterial.

TABLE 11 Stability of 150 mg/mL mAb-316P FDS¹ under stress conditionsStress No 45° C. 37° C. 25° C. Freeze/ Test Stress² Agitation IncubationIncubation Incubation Thaw Time of 0 60 120 14 28 14 28 14 28 8 Stressmin min min days days days days days days cycles Visual Pass Pass PassPass Pass Pass Pass Pass Pass Pass Appearance Turbidity³ 0.00 0.00 0.000.02 0.03 0.00 0.01 0.00 0.00 0.00 pH 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.06.0 6.0 % Total 100 101 102 98 98 99 98 100 99 101 Recovered % Native97.4 97.5 97.2 94.2 92.4 95.7 95.1 96.7 96.2 97.1 Recovered % Aggregate1.6 1.7 2.1 3.4 4.1 2.4 2.6 2.0 2.2 1.7 Recovered % Main 53.6 53.7 54.738.7 29.0 46.3 39.5 51.5 49.1 53.9 Recovered % Acidic 27.2 26.1 25.939.5 48.6 31.9 36.9 27.8 28.9 26.5 Recovered % Basic 19.3 20.2 19.5 21.822.5 21.8 23.7 20.7 22.0 19.6 Recovered Bioassay (% 84 NP 84 NP 85 NP 82NP 98 81 Relative Potency)⁴ ¹10 mM histidine, pH 6.0, 0.2% polysorbate20, 10% sucrose, 150 mg/mL anti-PCSK9 mAb-316P ²‘No stress’ values areaverages from both stability studies. ³Turbidity is reported as therelative change in OD at 405 nm as compared to the starting material⁴Percent relative potency; Acceptance criteria: 50-200% of referencestandard

TABLE 12 Stability of 175 mg/mL mAb-316P FDS¹ under stress conditionsStress No 45° C. 37° C. 25° C. Freeze/ Test Stress² Agitation IncubationIncubation Incubation Thaw Time of 0 60 120 14 28 14 28 14 31 8 Stressmin min min days days days days days days cycles Visual Pass Pass PassPass Pass Pass Pass NA Pass Pass Appearance Turbidity³ 0.00 0.00 0.010.01 0.03 0.00 0.01 NA 0.00 0.01 pH 6.0 6.0 6.0 6.1 6.1 6.0 6.0 NA 6.06.0 % Total 100 96 98 101 98 100 96 NA 99 98 Recovered % Native 96.596.3 96.4 94.5 91.7 95.7 94.7 NA 96.2 96.1 Recovered % Aggregate 2.5 2.52.5 3.6 5.2 2.8 3.0 NA 2.6 2.3 Recovered % Main 59.5 58.3 59.0 47.1 38.456.3 46.9 NA 58.2 59.3 Recovered % Acidic 30.1 29.3 29.4 39.5 47.6 32.339.8 NA 30.9 29.5 Recovered % Basic 10.3 12.4 11.6 13.4 14.1 11.4 13.4NA 11.0 11.1 Recovered Bioassay (% 0.00 0.00 0.01 0.01 0.03 0.00 0.01 NA0.00 0.01 Relative Potency)⁴ ¹10 mM histidine, pH 6.0, 0.01% polysorbate20, 5% sucrose, 50 mM arginine 175 mg/mL anti-PCSK9 mAb-316P. ²‘Nostress’ values are averages from both stability studies. ³Turbidity =change in OD at 405 nm relative to starting material. ⁴Percent relativepotency; Acceptance criteria: 50-200% of reference standard

TABLE 13 Stability of 150 mg/mL mAb-316P DP in PFS Storage 5° C. 25° C.45° C. 0 at 6 mo. at 6 mo. at 28 days. Appearance Pass Pass Pass PassTurbidity¹ 0.00 0.00 0.00 0.02 pH 6.1 6.1 6.1 6.1 % Total Recovered 100102 102 97 % Native Recovered 96.6 96.1 94.2 92.4 % Aggregate Recovered2.4 2.6 3.2 4.3 % Main Recovered 58.4 58.5 45.7 35.8 % Acidic Recovered31.8 31.3 42.1 50.9 % Basic Recovered 9.8 10.2 12.2 13.4 ¹Turbidity =change in OD at 405 nm relative to starting material.

TABLE 14 Stability of 175 mg/mL mAb-316P DP in PFS Storage 5° C. 25° C.45° C. 0 at 6 mo. at 6 mo. at 28 days. Appearance Pass Pass Pass PassTurbidity¹ 0.00 0.00 0.02 0.04 pH 6.1 6.1 6.1 6.1 % Total Recovered 100103 101 100 % Native Recovered 96.7 96.3 94.6 91.6 % Aggregate Recovered2.3 2.4 3.0 5.4 % Main Recovered 59.1 59.7 47.1 37.7 % Acidic Recovered31.2 30.6 40.6 48.2 % Basic Recovered 9.7 9.7 12.3 14.2 ¹Turbidity =change in OD at 405 nm relative to starting material.

TABLE 15 Compatibility of 150 mg/mL mAb-316P¹ for 14 Days at 40° C. NoStorage, Poly- Poly- Poly- Stainless Storage Glass Glass carbonatepropylene styrene Steel Turbidity² 0.00 0.00 0.00 0.01 0.01 0.01 pH 6.06.1 6.0 6.0 6.0 6.0 % Total Recovered 100 97 104 99 105 104 % NativeRecovered 97.4 95.9 95.8 95.7 95.7 95.5 % Agg'ate Recvr'd 1.7 2.5 2.62.7 2.6 2.7 % Main Recovered 53.3 45.8 45.8 44.7 45.6 45.1 % AcidicRecovered 26.9 33.0 32.9 33.8 33.0 33.5 % Basic Recovered 19.8 21.3 21.621.6 21.5 21.4 ¹10 mM Histidine, pH 6.0, 0.2% Polysorbate 20, 10%Sucrose, and 150 mg/mL mAb ²Turbidity = OD at 405 nm relative to thestarting material.

TABLE 16 Compatibility of 175 mg/mL mAb-316P¹ for 14 Days at 40° C. NoStorage, Poly- Poly- Poly- Stainless Storage Glass Glass carbonatepropylene styrene Steel Turbidity³ 0.00 0.00 0.00 0.00 0.00 0.01 pH 6.16.1 6.1 6.0 6.1 6.1 % Total Recovered 100 98 104 100 105 98 % NativeRecovered 96.6 95.3 95.2 95.1 95.2 94.9 % Agg'ate Recvr'd 2.4 3.0 3.13.2 3.1 3.3 % Main Recovered 57.7 51.3 51.0 50.6 51.0 50.4 % AcidicRecovered 30.0 34.5 34.5 35.0 34.5 35.2 % Basic Recovered 12.3 14.2 14.514.4 14.4 14.4 ¹10 mM Histidine, pH 6.0, 0.01% Polysorbate 20, 5%Sucrose, 50 mM arginine, and 175 mg/mL mAb ²Turbidity = OD at 405 nmrelative to the starting material.

TABLE 17 Charge Heterogeneity of mAb-316P by cIEF Lot 1 Lot 2 Lot 1:Lot2 (1:1 mix) Peak Peak Peak Peak No. pl Area, % pl Area, % pl Area, % 17.99 (0.01) 0.8 (0.1) 7.99 (0.01) 0.8 (0.0) 7.98 (0.01)  0.7 (0.1) 28.15 (0.00) 2.6 (0.1) 8.15 (0.01) 2.6 (0.1) 8.14 (0.01)  2.6 (0.1) 38.29 (0.00) 6.9 (0.1) 8.29 (0.00) 7.0 (0.0) 8.28 (0.01)  6.9 (0.1) 48.42 (0.00) 18.4 (0.2)  8.42 (0.00) 18.6 (0.2)  8.42 (0.01) 18.5 (0.1) 58.54 (0.01) 66.4 (0.3)  8.55 (0.01) 68.0 (0.3)  8.54 (0.00) 67.3 (0.1) 68.65 (0.00) 4.0 (0.1) 8.65 (0.00) 2.8 (0.1) 8.64 (0.01)  3.4 (0.0) 78.82 (0.00) 0.9 (0.1) 8.81 (0.00) 0.3 (0.1) 8.81 (0.01)  0.6 (0.1)

TABLE 18 mAb-316P glycosylated peptides Heavy Peak Chain Observed mass(Da) No. Fragment Lot 1 Lot 2 Comments 15a 294-302 2957.15 2957.19(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₂(Gal)₂ 294-302 2795.11 2795.11(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₂(Gal)₁ 294-302 2404.94 2404.94(GlcNAc)₂(Man)₅ 294-302 2592.02 2591.96(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₁(Gal)₁ 294-302 2267.94 2267.92(Fuc)₁(GlcNAc)₂(Man)₂(GlcNAc)₁ 294-302 2121.82 2121.80(GlcNAc)₂(Man)₂(GlcNAc)₁ 15b 294-302 2283.91 2283.91(GlcNAc)₂(Man)₃(GlcNAc)₁ 294-302 2429.98 2429.98(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₁ 294-302 2486.99 2486.97(GlcNAc)₂(Man)₃(GlcNAc)₂ 294-302 2633.05 2633.06(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₂ 16 294-302 1188.52 1188.53non-glycosylated NST site 17a 290-302 3439.44 3439.44(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₂(Gal)₂ 290-302 3278.40 3277.35(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₂(Gal)₁ 290-302 2887.20 2887.26(GlcNAc)₂(Man)₅ 17b 290-302 2766.24 2766.21 (GlcNAc)₂(Man)₃(GlcNAc)₁290-302 2969.34 2969.25 (GlcNAc)₂(Man)₃(GlcNAc)₂ 290-302 2912.28 2912.25(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₁ 290-302 3115.32 3115.35(Fuc)₁(GlcNAc)₂(Man)₃(GlcNAc)₂

TABLE 19 Integrated Peak Areas of Glycans Identified by CapillaryElectrophoresis 1:1 lot Lot 1 Peak Lot 2 Peak mixture Peak Glycan GlycanArea^(a), % Area, % Area, % Identity¹ Structure² 1  8.6 (0.2)  6.8 (0.1) 7.6 (0.2) G0-Fuc

2  1.8 (0.0)  2.9 (0.1)  2.3 (0.1) Man5

3  0.7 (0.0)  0.5 (0.0)  0.6 (0.0) Minor peak is unidentified 4 BelowLOQ Below LOQ Below LOQ Minor peak is unidentified 5 Below LOQ Below LOQBelow LOQ Minor peak is unidentified 6 34.4 (0.1) 45.8 (0.2) 40.4 (0.2)G0

7  1.5 (0.0)  0.7 (0.0)  1.1 (0.0) Minor peak is unidentified 8 BelowLOQ  0.9 (0.0)  0.6 (0.0) Minor peak is unidentified 9 Below LOQ  0.7(0.0)  0.6 (0.0) Minor peak is unidentified 10 29.5 (0.1) 24.7 (0.0)27.0 (0.1) G1(1-6)

11 10.1 (0.1)  8.6 (0.0)  9.3 (0.1) G1(1-3) and/or G2-Fuc

12 Below LOQ Below LOQ Below LOQ Minor peak is unidentified 13 Below LOQBelow LOQ Below LOQ Minor peak is unidentified 14 11.7 (0.0)  7.1 (0.1) 9.4 (0.2) G2

¹G#-fuc and G# refer to non-fucosylated and fucosylated glycans,respectively. The symbol # refers to 0, 1, or 2. ²Glycan Key:

fucose,

N-acetyl glucosamine,

mannose,

galactose. LOQ = Limit of Quantification.

What is claimed is:
 1. A pre-filled syringe composition comprising aliquid pharmaceutical formulation, wherein the liquid pharmaceuticalformulation comprises: (a) 50±7.5 mg/mL to 250±37.5 mg/mL of an antibodyor antigen-binding fragment thereof that specifically binds humanproprotein convertase subtilisin kexin-9 (PCSK9), wherein the antibodycomprises a heavy chain complementarity determining region (HCDR) 1 ofSEQ ID NO:2, an HCDR2 of SEQ ID NO:3, an HCDR3 of SEQ ID NO:4, a lightchain complementarity determining region (LCDR) 1 of SEQ ID NO:6, anLCDR2 of SEQ ID NO:7, and an LCDR3 of SEQ ID NO:8; (b) 10±1.5 mMhistidine (pH 6.0±0.3); (c) 0.01±0.0015% w/v polysorbate 20; and (d)10±1.5% sucrose; wherein said liquid pharmaceutical formulation iscontained within a syringe.
 2. The pre-filled syringe composition ofclaim 1, wherein the syringe is a normal tungsten syringe.
 3. Thepre-filled syringe composition of claim 1, wherein the syringe is a lowtungsten syringe.
 4. The pre-filled syringe composition of claim 1,wherein the syringe comprises a coated plunger.
 5. The pre-filledsyringe composition of claim 4, wherein the coated plunger is coatedwith a fluorocarbon film.
 6. The pre-filled syringe composition of claim1, wherein the syringe is a low tungsten syringe, and wherein thesyringe comprises a coated plunger.
 7. The pre-filled syringecomposition of claim 1, wherein the syringe is a 1 mL long glass syringecomprising a 27 gauge thin wall needle, a fluorocarbon coated rubberplunger, and a rubber needle shield.
 8. The pre-filled syringecomposition of claim 1, wherein the antibody comprises a heavy chainvariable domain (HCVD) comprising SEQ ID NO:1, and a light chainvariable domain (LCVD) comprising SEQ ID NO:5.
 9. The pre-filled syringecomposition of claim 1, wherein: over 90% of the antibodies have amolecular weight of 155 kDa±1 kDa; over 50% of the antibodies have anisoelectric point of about 8.5; and from 75% to 90% of the antibodiesare fucosylated.
 10. The pre-filled syringe composition of claim 1,wherein at least 91% of the antibody has native conformation after 28days at 45° C.
 11. The pre-filled syringe composition of claim 1,wherein at least 35% of the antibody is the main charge variant of theantibody after 28 days at 45° C.
 12. The pre-filled syringe compositionof claim 1, wherein at least 94% of the antibody has native conformationafter six months at 25° C.
 13. The pre-filled syringe composition ofclaim 1, wherein at least 45% of the antibody is the main charge variantof the antibody after six months at 25° C.
 14. The pre-filled syringecomposition of claim 1, wherein at least 96% of the antibody has nativeconformation after six months at 5° C.
 15. The pre-filled syringecomposition of claim 1, wherein at least 58% of the antibody is the maincharge variant of the antibody after six months at 5° C.
 16. Thepre-filled syringe composition of claim 1, wherein at least 96% of theantibody is the main charge variant of the antibody after three monthsat −20° C., −30° C., or −80° C.
 17. The pre-filled syringe compositionof claim 1, wherein at least 56% of the antibody is the main chargevariant of the antibody after three months at −20° C., −30° C., or −80°C.
 18. The pre-filled syringe composition of claim 1, wherein theformulation comprises about 175 mg/mL of the antibody.
 19. Thepre-filled syringe composition of claim 1, wherein the formulationcomprises about 150 mg/mL of the antibody.
 20. The pre-filled syringecomposition of claim 1, wherein the formulation comprises about 100mg/mL of the antibody.
 21. The pre-filled syringe composition of claim1, wherein the formulation comprises about 75 mg/mL of the antibody. 22.The pre-filled syringe composition of claim 1, wherein the formulationcomprises about 50 mg/mL of the antibody.